U.S. patent application number 13/474980 was filed with the patent office on 2012-10-11 for rack for sample tubes and reagent holders.
This patent application is currently assigned to HandyLab, Inc.. Invention is credited to Patrick Duffy, Kalyan Handique, Jeff Williams, Kerry Wilson.
Application Number | 20120258463 13/474980 |
Document ID | / |
Family ID | 40669882 |
Filed Date | 2012-10-11 |
United States Patent
Application |
20120258463 |
Kind Code |
A1 |
Duffy; Patrick ; et
al. |
October 11, 2012 |
RACK FOR SAMPLE TUBES AND REAGENT HOLDERS
Abstract
A rack for holding samples and various reagents, wherein the
rack may be used for loading the samples and reagents prior to
using the reagents. The rack accepts complementary reagent holders,
each of which contain a set of reagents for carrying out a
predetermined processing operation, such as preparing biological
samples for amplifying and detecting polynucleotides extracted from
the samples.
Inventors: |
Duffy; Patrick; (Whitmore
Lake, MI) ; Wilson; Kerry; (Elkhart, IN) ;
Handique; Kalyan; (Ypsilanti, MI) ; Williams;
Jeff; (Chelsea, MI) |
Assignee: |
HandyLab, Inc.
Ann Arbor
MI
|
Family ID: |
40669882 |
Appl. No.: |
13/474980 |
Filed: |
May 18, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12178584 |
Jul 23, 2008 |
8182763 |
|
|
13474980 |
|
|
|
|
12173023 |
Jul 14, 2008 |
8133671 |
|
|
12178584 |
|
|
|
|
12218498 |
Jul 14, 2008 |
|
|
|
12178584 |
|
|
|
|
60959437 |
Jul 13, 2007 |
|
|
|
60959437 |
Jul 13, 2007 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
422/547; 422/549; 422/561; 422/562 |
Current CPC
Class: |
B01L 2200/16 20130101;
G01N 35/026 20130101; G01N 35/0099 20130101; G01N 2035/0493
20130101; B01L 9/527 20130101; F16K 99/0044 20130101; B01L 7/52
20130101; F16K 99/0061 20130101; B01L 3/5082 20130101; B03C 1/01
20130101; F16K 99/003 20130101; G01N 35/1002 20130101; F16K 99/0032
20130101; B03C 1/30 20130101; C12N 15/1003 20130101; B01L 9/06
20130101; B03C 2201/26 20130101; Y10T 436/113332 20150115; F16K
2099/0084 20130101; F16K 99/0001 20130101; G01N 2035/00881
20130101; G01N 2035/00772 20130101; B03C 1/0332 20130101; B03C
1/288 20130101; G01N 2035/00801 20130101; B03C 2201/18
20130101 |
Class at
Publication: |
435/6.12 ;
422/561; 422/562; 422/547; 422/549 |
International
Class: |
B01L 9/00 20060101
B01L009/00; B01L 9/06 20060101 B01L009/06; C12Q 1/68 20060101
C12Q001/68 |
Claims
1. An apparatus for holding reagents for sample preparation,
comprising: a horizontal member comprising a plurality of slots
arranged parallel to an axis between a first side and a second side
of the horizontal member, each of the plurality of slots defining
an opening in the first side of the horizontal member and extending
toward the second side of the horizontal member, each of the
plurality of slots configured to accept a reagent holder; and two
or more members supporting the horizontal member.
2. The apparatus of claim 1, further comprising a plurality of
sample tube holders, wherein each of the plurality of sample tube
holders corresponds to a single slot.
3. The apparatus of claim 2, wherein each of the plurality of
sample tube holders is arranged in line with a single slot.
4. The apparatus of claim 2, wherein each of the sample tube
holders comprises a recessed portion configured to receive a bottom
of a sample tube, and an aperture configured to hold an upper
portion of the sample tube.
5. The apparatus of claim 1, further comprising a reagent holder
located in at least one of the plurality of slots, the reagent
holder comprising a process container and one or more reagent
receptacles.
6. The apparatus of claim 5, wherein the reagent holder contains
sufficient reagents to extract nucleic acids from a sample.
7. The apparatus of claim 5, wherein the reagent holder further
comprises at least one pipette tip holder.
8. The apparatus of claim 5, wherein the apparatus is configured to
be removably received by a heater unit configured to independently
heat the process chamber of each reagent holder located in the
apparatus.
9. The apparatus of claim 1, further comprising a sensor configured
to indicate proper placement of the apparatus in a diagnostic
apparatus.
10. The apparatus of claim 1, wherein the apparatus is configured
to be removably oriented in a receiving bay of a diagnostic
apparatus.
11. The apparatus of claim 1, wherein the plurality of slots
consists of 2, 5, 6, 8, 10, 12, 16, 20, or 24 openings.
12. The apparatus of claim 1, further comprising an identifier
associated with each slot.
13. The apparatus of claim 1, further comprising a reagent holder
located in each of the plurality of slots.
14. A housing for samples and reagent holders, comprising: a top
surface comprising a plurality of parallel lanes extending across
the top surface, each lane defining an axis upon which a first
opening configured to receive a reagent holder and a second opening
configured to receive a sample tube are aligned; at least two
surfaces supporting the top surface; and a reagent holder located
in the first opening of at least one of the plurality of lanes, the
reagent holder comprising a process container and one or more
reagent receptacles.
15. The housing of claim 14, further comprising a sample tube
located in the second opening of at least one of the plurality of
lanes.
16. The housing of claim 14, wherein each lane defines an opening
in a first side of the top surface and extends along the axis
toward a second side of the top surface.
17. The housing of claim 14, wherein the reagent holder contains
sufficient reagents to extract nucleic acids from a sample.
18. The housing of claim 14, wherein the housing is configured to
be removably received by a heater unit configured to independently
heat the process chamber of each reagent holder located in the
housing.
19. A method of preparing a plurality of samples for extraction of
nucleic acid contained in the samples, the plurality of samples
held in a housing comprising a top surface comprising a plurality
of slots arranged parallel to an axis between a first side and a
second side of the top surface, each of the plurality of slots
defining an opening in the first side of the top surface and
extending toward the second side of the top surface, the method
comprising: sliding a first reagent holder through the opening of a
first slot of the plurality of slots and toward the second side of
the top surface; inserting a first sample tube containing a first
sample into a first sample tube holder arranged in line with the
first slot; sliding a second reagent holder through the opening of
a second slot of the plurality of slots and toward the second side
of the top surface; inserting a second sample tube containing a
second sample into a second sample tube holder arranged in line
with the second slot; and inserting the housing into a receiving
bay of a diagnostic apparatus configured to extract nucleic acid
from the first sample and the second sample independently.
20. The method of claim 19, wherein the first reagent holder and
the second reagent holder comprise a process container and one or
more reagent receptacles containing reagents.
21. The method of claim 20, further comprising dispensing the first
sample into one or more reagent receptacles of the first reagent
holder.
22. The method of claim 20, further comprising inserting the
housing into a heater unit configured to independently heat the
process chamber of the first reagent holder and the second reagent
holder.
Description
CLAIM OF PRIORITY
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/178,584, filed Jul. 23, 2008, which is a
continuation-in-part of U.S. patent application Ser. No.
12/173,023, filed Jul. 14, 2008, issued as U.S. Pat. No. 8,133,671
on Mar. 13, 2012, which claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Application No. 60/959,437, filed
Jul. 13, 2007. U.S. patent application Ser. No. 12/178,584, filed
Jul. 23, 2008, is a continuation-in-part of U.S. patent application
Ser. No. 12/218,498, filed Jul. 14, 2008, which claims priority
under 35 U.S.C. .sctn.119(e) to U.S. Provisional Application No.
60/959,437, filed Jul. 13, 2007. The disclosures of all of the
above-referenced prior applications, publications, and patents are
considered part of the disclosure of this application, and are
incorporated by reference herein in their entirety.
TECHNICAL FIELD
[0002] The technology described herein generally relates to racks
for holding samples and various reagents, wherein the rack may be
used for loading the samples and reagents prior to using the
reagents. The technology more particularly relates to racks that
accept complementary reagent holders each of which contain a set of
reagents for carrying out a predetermined processing operation,
such as preparing biological samples for amplifying and detecting
polynucleotides extracted from the samples.
BACKGROUND
[0003] The medical diagnostics industry is a critical element of
today's healthcare infrastructure. At present, however, diagnostic
analyses no matter how routine have become a bottleneck in patient
care. There are several reasons for this. First, many diagnostic
analyses can only be done with highly specialist equipment that is
both expensive and only operable by trained clinicians. Such
equipment is found in only a few locations--often just one in any
given urban area. This means that most hospitals are required to
send out samples for analyses to these locations, thereby incurring
shipping costs and transportation delays, and possibly even sample
loss or mishandling. Second, the equipment in question is typically
not available `on-demand` but instead runs in batches, thereby
delaying the processing time for many samples because they must
wait for a machine to fill up before they can be run.
[0004] Understanding that sample flow breaks down into several key
steps, it would be desirable to consider ways to automate as many
of these as possible. For example, a biological sample, once
extracted from a patient, must be put in a form suitable for a
processing regime that typically involves using PCR to amplify a
vector of interest. Once amplified, the presence of a nucleotide of
interest from the sample needs to be determined unambiguously.
Preparing samples for PCR is currently a time-consuming and labor
intensive step, though not one requiring specialist skills, and
could usefully be automated. By contrast, steps such as PCR and
nucleotide detection have customarily only been within the compass
of specially trained individuals having access to specialist
equipment.
[0005] Sample preparation is labor intensive in part because of the
number of reagents required, and the need for multiple liquid
transfer (e.g., pipetting) operations. Furthermore, the reagents
required are of sufficient variety that they typically require
different handling from one another and are available from
different vendors. Even where reagents can be collected together in
a single holder and made ready for use, such as described in
copending application Ser. No. 12/______, filed by ExpressMail on
Jul. 14, 2008 (and entitled "Reagent Tube, Reagent Holder, and Kits
Containing Same", in the name of Wilson, et al.) and incorporated
herein by reference, it would be beneficial to be able to set up a
number of holders for use in batches, and to make them available to
liquid dispensing tools that can operate on holders in
parallel.
[0006] The discussion of the background herein is included to
explain the context of the inventions described herein. This is not
to be taken as an admission that any of the material referred to
was published, known, or part of the common general knowledge as at
the priority date of any of the claims.
[0007] Throughout the description and claims of the specification
the word "comprise" and variations thereof, such as "comprising"
and "comprises", is not intended to exclude other additives,
components, integers or steps.
SUMMARY
[0008] A rack, comprising: a housing; a plurality of lanes in the
housing, and wherein each lane of the plurality of lanes comprises:
a first location configured to accept a sample tube; and a second
location, configured to accept a reagent holder; and a registration
member complementary to a receiving bay of a diagnostic
apparatus.
[0009] A rack, wherein the registration member comprises two or
more positioning pins configured to: ensure that the rack can only
be placed in the diagnostic apparatus in a single orientation; and
provide stability for the rack when placed in the diagnostic
apparatus.
[0010] A rack configured to receive a plurality of reagent holders,
and to receive a plurality of sample tubes, wherein the reagent
holders are in one-to-one correspondence with the sample tubes, and
wherein the reagent holders each contain sufficient reagents to
extract polynucleotides from the sample and place the
polynucleotides into a PCR-ready form, and wherein the rack is
configured to be insertable into, and removable from, an apparatus
that carries out PCR on the PCR-ready polynucleotides.
[0011] A method of carrying out sample preparation on multiple
samples in parallel, wherein reagents for carrying out preparation
on each sample are contained in respective reagent holders, the
holders being supported by a rack, as further described herein. The
method, can be carried out automatically by a diagnostic apparatus
in which the rack is situated, or can be carried out manually, when
the rack is positioned on a horizontal surface, e.g., a laboratory
benchtop.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows perspective views of an exemplary rack for
samples and reagent holders.
[0013] FIG. 2 shows perspective views of a heater unit and the rack
of FIG. 1 positioned in conjunction with the heater unit.
[0014] FIG. 3 shows a perspective view of an exemplary rack for
samples and reagent holders.
[0015] FIGS. 4A-4K show various views of the rack of FIG. 3.
[0016] FIG. 5 shows an area of a diagnostic apparatus configured to
accept a rack of FIG. 3.
[0017] FIG. 6 shows a perspective view of an exemplary rack,
carrying a number of sample tubes and reagent holders, and
positioned in a diagnostic apparatus.
[0018] FIGS. 7A and 7B show an exemplary embodiment of a reagent
holder having a pipette sheath, in perspective view (FIG. 7A) and
underside view (FIG. 7B).
[0019] FIG. 8 shows a schematic of an automated apparatus
configured to carry out sample preparation using a rack as
described herein.
[0020] Like reference numerals in the various drawings indicate
like elements.
DETAILED DESCRIPTION
[0021] Described herein are racks for supporting, carrying, and
transporting reagents for various purposes, in particular in
connection with sample preparation in a clinical context. The rack
permits placement of one or more sample tubes and one or more
corresponding holders of reagents, such as analyte specific
reagents (ASR's) and/or sample preparation reagents. The samples
and holders, when so placed, are positioned to carry out liquid
dispensing processes, associated with sample preparation such as
for PCR, that minimize cross-sample contamination but permit
multiple PCR preparations to be performed from multiple clinical
samples, in batches, or in parallel. The racks are configured for
use in an automated preparatory apparatus that can carry out sample
preparation on samples in more than one holder positioned in a rack
simultaneously.
[0022] The racks as described herein find particular application to
analyzing any nucleic acid containing sample for any purpose,
including but not limited to genetic testing, and clinical testing
for various infectious diseases in humans.
[0023] In various embodiments, preparation of a PCR-ready sample
for use in subsequent diagnosis, can include one or more of the
following steps: contacting a neutralized polynucleotide sample
with a PCR reagent mixture comprising a polymerase enzyme and a
plurality of nucleotides (in some embodiments, the PCR reagent
mixture can further include a positive control plasmid and a
fluorogenic hybridization probe selective for at least a portion of
the plasmid). In some embodiments, the PCR reagent mixture can be
in the form of one or more lyophilized pellets, as stored in a
container on a reagent holder, and the method can further include
reconstituting the PCR pellet with liquid to create a PCR reagent
mixture solution. The holders configured for use with racks as
described herein provide, in a self-contained manner, all of the
reagents required to prepare a PCR-ready sample, or, when delivered
to a user in kit form, contain in conjunction with other packages
all of the required reagents. Suitable reagents, and protocols for
using the same in DNA and RNA extractions can be found in,
respectively, copending application Ser. Nos. 12/172,208, and
12/172,214, both filed Jul. 11, 2008 and incorporated herein by
reference.
[0024] The racks herein are typically configured for use by an
apparatus that carries out automated sample preparation, for
example, on multiple samples simultaneously. An exemplary form of
such an apparatus is further described herein, and can also be
found described in U.S. provisional Patent Application Ser. No.
60/959,437, filed Jul. 13, 2007, incorporated herein by reference
in its entirety, and in U.S. patent application Ser. No.
12/173,023, filed on Jul. 14, 2008 (and entitled "Integrated
Apparatus for Performing Nucleic Acid Extraction and Diagnostic
Testing on Multiple Biological Samples", in the name of Williams,
et al.), incorporated by reference herein.
Rack
[0025] The racks herein are typically configured to be insertable
into, and removable from, a diagnostic or preparatory apparatus,
each of the racks being further configured to receive a plurality
of reagent holders, and to receive a plurality of sample tubes,
wherein the reagent holders are in one-to-one correspondence with
the sample tubes, and wherein the reagent holders each contain
sufficient reagents to extract polynucleotides from a sample and
place the polynucleotides into a PCR-ready form. Exemplary reagent
holders are further described elsewhere herein and also in
copending application Ser. No. 12/______, filed by ExpressMail on
Jul. 14, 2008 (and entitled "Reagent Tube, Reagent Holder, and Kits
Containing Same", in the name of Wilson, et al.) and incorporated
herein by reference. An exemplary apparatus is outlined herein, and
also described in U.S. patent application Ser. No. 12/173,023,
filed on Jul. 14, 2008 (and entitled "Integrated Apparatus for
Performing Nucleic Acid Extraction and Diagnostic Testing on
Multiple Biological Samples", in the name of Williams, et al.),
incorporated by reference herein.
[0026] Two perspective views of an exemplary rack 800, configured
to accept 12 sample tubes and 12 corresponding reagent holders, in
12 lanes 850, are shown in FIG. 1. A lane 850, as used herein in
the context of a rack, is a dedicated region of the rack designed
to receive a sample tube and corresponding reagent holder. In the
accompanying drawings, not all visible lanes are labeled; usually
one lane or a pair of lanes in a given view is given reference
number 850. Two perspective views of the same exemplary rack, in
conjunction with a heater unit, as further described herein, are
shown in FIG. 2.
[0027] A rack may accept 2, 4, 6, 8, 10, 12, 16, or 20 samples such
as in sample tubes 802, and a corresponding number of holders 804.
Thus the embodiment of FIG. 1 configured to receive 12 samples and
12 corresponding reagent holders is exemplary.
[0028] A rack is typically configured to accept a number of reagent
holders 804, such as those further described herein, the rack being
configured to hold one or more such holders, either permitting
access on a laboratory benchtop to reagents stored in the holders,
or situated in a dedicated region of the apparatus permitting the
holders to be accessed by one or more other functions of the
apparatus, such as automated pipetting, heating of the process
tubes, and magnetic separating of affinity beads. Typically holders
804 each have at least one or more pipette tips, and one or more
containers for reagents.
[0029] Various views of a second exemplary rack 800, also
configured to accept 12 sample tubes and 12 reagent holders, are
shown in FIG. 3, and FIGS. 4A-4K. Thus, the following views are
shown: side plan (FIG. 4A); front plan, showing sample tubes (FIG.
4B); rear plan, showing reagent holders (FIG. 4C); rear elevation,
showing reagent holders (FIG. 4D); front elevation, showing sample
tubes (FIG. 4E); top, showing insertion of a reagent holder (FIGS.
4F and 4G); top showing slot for inserting a reagent holder (FIG.
411); top view showing registration of reagent holder (FIG. 41);
close up of rack in state of partial insertion/removal from
apparatus (FIG. 4J); and rack held by handle, removed from
apparatus (FIG. 4K).
[0030] In FIG. 4J, two racks are visible: rack 800-1 is shown (in
part) in position inserted fully into an apparatus 981; rack 800-2
is shown in a state of partial insertion or removal from apparatus
981. A recessed area, such as a receiving bay, in a diagnostic or
preparatory apparatus 981, as further described herein, for
accepting the exemplary removable rack of FIG. 3 is shown in FIG.
5. A rack, as shown in FIG. 3, situated in the recessed area of
FIG. 5 is shown in FIG. 6, in location, in relation to a receiving
bay 992 holding a microfluidic cartridge 994 that can be configured
to carry out an amplification on a suitably prepared sample, as
further described in U.S. patent application Ser. No. 12/173,023,
filed on Jul. 14, 2008 (and entitled "Integrated Apparatus for
Performing Nucleic Acid Extraction and Diagnostic Testing on
Multiple Biological Samples", in the name of Williams, et al.).
Other suitably configured recessed areas for receiving other racks
differing in shape, appearance, and form, rather than function, are
consistent with the description herein.
[0031] The two exemplary racks shown in FIGS. 1 and 3 being
non-limiting, general features of racks contemplated herein are now
described using the two exemplary racks as illustrative thereof.
For example, in the embodiments shown here, at least the first lane
and the second lane are parallel to one another, a configuration
that increases pipetting efficiency. Typically, when parallel to
one another, pairs of adjacent sample lanes 850 are separated by 24
mm at their respective midpoints. Other distances are possible,
such as 18 mm apart, or 27 mm apart. The distance between the
midpoints in dependent on the pitch of the nozzles in the liquid
dispensing head, as further described herein. Keeping the spacing
in multiples of 9 mm enables easy loading from the rack into a 96
well plate (where typically wells are spaced apart by 9 mm).
Typically, also, the rack is such that the plurality of reagent
holders in the plurality of lanes are maintained at the same height
relative to one another.
[0032] The rack is configured to accept a given reagent holder in
such a way that the reagent holder snaps or locks reversibly into
place, and thereby remains steady while reagents are accessed in
it, and while the rack is being carried from one place to another
or is being inserted into, or removed from, a diagnostic
apparatus.
[0033] In each embodiment of FIGS. 1 and 3, each of the second
locations in the respective lanes comprises a mechanical key
configured to accept the reagent holder in a single orientation. In
FIG. 1, it is shown that the reagent holder(s) slide horizontally
into vertically oriented slots, one per holder, located in the
rack. In such an embodiment, the edge of a connecting member on the
holder engages with a complementary groove in the upper portion of
a slot. In FIGS. 4F, 4G, and 4I, it is shown that the reagent
holder(s) can engage with the rack via a mechanical key that keeps
the holders steady and in place. For example, the mechanical key
can comprise a raised or recessed portion that, when engaging with
a complementary portion of the reagent holder, permits the reagent
holder to snap into the second location. It can also be seen in the
embodiments shown that the reagent holder has a first end and a
second end, and the mechanical key comprises a first feature
configured to engage with the first end, and a second feature
configured to engage with the second end in such a way that a
reagent holder cannot be inserted into the rack the wrong way
around. In some embodiments, as shown in FIG. 8H, a sprung metal
tab 833 positioned inside the slot serves to keep the holder
tightly in place against a stop at the opposite end of the
slot.
[0034] In certain embodiments the reagent holders each lock into
place in the rack, such as with a cam locking mechanism that is
recognized as locked audibly and/or physically, or such as with a
mechanical key.
[0035] The rack can be configured so that the holders, when
positioned in it, are aligned for proper pipette tip pick-up using
a liquid dispenser as further described herein. Furthermore, the
second location of each lane can be deep enough to accommodate one
or more pipette tips, such as contained in a pipette tip
sheath.
[0036] In certain embodiments, the rack is configured to accept the
samples in individual sample tubes 802, each mounted adjacent to a
corresponding holder 804, for example on one side of rack 800. The
sample tubes can be accessible to a sample identification verifier
such as a bar code reader, as further described herein. In FIG. 1,
a sample tube is held at its bottom by a cylindrical receiving
member. In FIG. 3, it is shown that a sample tube can be held at
both its top and bottom, such as by a recessed portion 803
configured to receive a bottom of a sample tube, and an aperture
805 configured to hold an upper portion of the sample tube. The
aperture can be a ring or an open loop, or a hole in a metal sheet.
The recessed portion can be as in FIG. 3, wherein it is an angled
sheet of metal housing having a hole large enough to accommodate a
sample tube. In alternate embodiments, a sample tube may be held at
its lower portion by a member having an aperture and located above
a base member on which the bottom of the sample tube rests.
[0037] The rack can be designed so that it can be easily removed
from the apparatus and carried to and from the laboratory
environment external to the apparatus, such as a bench, and the
apparatus, for example, to permit easy loading of the sample
tube(s) and the reagent holder(s) into the rack. In certain
embodiments, the rack is designed to be stable on a horizontal
surface, and not easily toppled over during carriage, and, to this
end, the rack has one or more (such as 2, 3, 4, 6, 8) feet 809. In
certain embodiments, the rack has a handle 806 to ease lifting and
moving, and as shown in FIG. 1, the handle can be locked into a
vertical position, during carriage, also to reduce risk of the rack
being toppled over. The handle can optionally have a soft grip 808
in its middle. In the embodiment of FIG. 3, the carrying handle is
positioned about an axis displaced from an axis passing through the
center of gravity of the rack when loaded, and is free to fall to a
position flush with an upper surface of the rack, under its own
weight.
[0038] The embodiment of FIG. 1 has a metallic base member 810
having 4 feet 811 that also serve as position locators when
inserting the rack into the dedicated portion of the apparatus. The
handle is attached to the base member, but other attachments for a
handle are possible. The portion of the rack 812 that accepts the
samples and holders can be made of plastic, and comprises 12 slots,
and may be disposable.
[0039] In the embodiment of FIG. 3, the rack comprises a housing, a
plurality of lanes in the housing, and wherein each lane of the
plurality of lanes comprises: a first location 851 configured to
accept a sample tube; and a second location 852, configured to
accept a reagent holder; and a registration member complementary to
a receiving bay of a diagnostic apparatus. Typically, the housing
is made of a metal, such as aluminum, that is both light but also
can be machined to high tolerance and is sturdy enough to ensure
that the rack remains stable when located in the diagnostic
apparatus. The registration member in FIG. 3 comprises four (4)
tight tolerance pegs 815, located one per corner of the rack. Such
pegs are such that they fit snugly and tightly into complementary
holes in a receiving area, such as a recessed area, of the
apparatus and thereby stabilize the rack. Other embodiments having,
for example, 2, or 3, or greater than 4 such pegs are consistent
with the embodiments herein.
[0040] In particular, the housing in the embodiment of FIG. 3
comprises a horizontal member 821, and two or more vertical members
822 connected to the horizontal member, and is such that the second
location 852 of each respective lane is a recessed portion within
the horizontal member. The two or more vertical members 809 in the
embodiment of FIG. 3 are configured to permit the rack to free
stand thereon. The housing may further comprise two or more feet or
runners, attached symmetrically to the first and second vertical
members and giving the rack additional stability when positioned on
a laboratory bench top.
[0041] Furthermore, in the embodiment of FIG. 3, the housing
further comprises a plurality of spacer members 825, each of which
is disposed between a pair of adjacent lanes. Optionally, such
spacer members may be disposed vertically between the lanes.
[0042] Although not shown in the FIGs., a rack can further comprise
a lane identifier associated with each lane. A lane identifier may
be a permanent or temporary marking such as a unique number or
letter, or can be an RFID, or bar-code, or may be a colored tag
unique to a particular lane.
[0043] A rack is configured so that it can be easily placed at the
appropriate location in a diagnostic apparatus and gives the user
positive feedback, such as audibly or physically, that it is placed
correctly. In certain embodiments, the rack can be locked into
position. It is desirable that the rack be positioned correctly,
and not permitted to move thereafter, so that movement of the
liquid dispenser will not be compromised during liquid handling
operations. The rack therefore has a registration member to ensure
proper positioning. In the embodiment of FIG. 3, the registration
member comprises two or more positioning pins configured to ensure
that the rack can only be placed in the diagnostic apparatus in a
single orientation; and provide stability for the rack when placed
in the diagnostic apparatus. The embodiment of FIG. 3 has,
optionally, a sensor actuator 817 configured to indicate proper
placement of the rack in the diagnostic apparatus. Such a sensor
may communicate with a processor 980 to provide the user with a
warning, such as an audible warning, or a visual warning
communicated via an interface, if the rack is not seated correctly.
It may also be configured to prevent a sample preparation process
from initiating or continuing if a seating error is detected.
[0044] In certain embodiments, the interior of the rack around the
location of process tubes in the various holders is configured to
have clearance for a heater assembly and/or a magnetic separator as
further described herein. For example, the rack is configured so
that process chambers on the individual holders are accepted by
heater units in a heater assembly as further described herein.
[0045] Having a removable rack enables a user to keep a next rack
loaded with samples and in line while a previous rack of samples is
being processed by the apparatus, so that thereby the apparatus
usage time and throughput is maximized.
[0046] The rack can also be conveniently cleaned outside of the
instrument in case of any sample or reagent spills over it or just
as a routine maintenance of laboratory wares.
[0047] In certain embodiments the racks have one or more disposable
parts.
Reagent Holders
[0048] Described herein are reagent holders for holding and
transporting reagents for various purposes, in particular sample
preparation in a clinical context, and configured to be received by
a rack as described herein. The reagent holders also typically
provide a container in which various reagents can be mixed one with
another and/or with a sample. The reagent holders typically permit
snapping in of one or more tubes of analyte specific reagents
(ASR's) and/or sample preparation reagents, and carrying out liquid
dispensing processes, associated with sample preparation, that
minimize cross-sample contamination but permit multiple PCR
preparations to be performed from a single clinical sample. The
holders are also configured for use in an automated preparatory
apparatus that can carry out sample preparation on samples in more
than one holder simultaneously.
[0049] FIGS. 7A and 7B show views of an exemplary holder 804 as
further described herein. This exemplary holder, as well as others
consistent with the written description herein though not shown as
specific embodiments, are now described.
[0050] The exemplary holder shown in FIGS. 7A and 7B can also be
referred to as a "unitized disposable strip", or a "unitized
strip", because it is intended to be used as a single unit that is
configured to hold all of the reagents and receptacles necessary to
perform a sample preparation, and because it is laid out in a strip
format. It is consistent with the description herein, though, that
other geometric arrangements of the various receptacles of the
holder are contemplated, so that the description is not limited to
a linear, or strip, arrangement, but can include a circular or grid
arrangement. Accordingly, a rack as described herein can
alternatively be configured to accept reagent holders that adopt
other shapes than strips. Alternative shapes could be square,
rectangular, and circular.
[0051] The exemplary holder of FIG. 7A comprises a connecting
member 510 having one or more characteristics as follows.
Connecting member 510 serves to connect various components of the
holder together. Connecting member 510 has an upper side 512 and,
opposed to the upper side, an underside 514.
[0052] The reagent holder of FIG. 7A is configured to comprise: a
process tube 520 affixed to the connecting member and having an
aperture 522 located in the connecting member; at least one socket
530, located in the connecting member, the socket configured to
accept a disposable pipette tip 580; an optional pipette sheath 570
as further described herein; two or more reagent tubes 540 disposed
on the underside of the connecting member, each of the reagent
tubes having an inlet aperture 542 located in the connecting
member; and one or more receptacles 550, located in the connecting
member, wherein the one or more receptacles are each configured to
receive a complementary container such as a reagent tube (not
shown) inserted from the upper side 512 of the connecting member.
The lanes of the rack described herein are designed to have
sufficient depth and width to accommodate the various reagent
tubes, receptacles, process tube, and pipette sheath of a given
reagent holder.
[0053] In FIG. 7B, a view of underside 514 is shown, having various
struts 597 connecting a rim of the connecting member with variously
the sockets, process tube, and reagent tubes. Struts 597 are
optional, and may be omitted all or in part, or may be substituted
by, in all or in part, other supporting pieces that connect various
parts of the holder to one another.
[0054] The one or more receptacles 550 are configured to accept
reagent tubes that contain, respectively, sufficient quantities of
one or more reagents typically in solid form, such as in
lyophilized form, for carrying out extraction of nucleic acids from
a sample that is associated with the holder. The receptacles can be
all of the same size and shape, or may be of different sizes and
shapes from one another. Receptacles 550 are shown as having open
bottoms, but are not limited to such topologies, and may be closed
other than the inlet 552 in the upper side of connecting member
510. Preferably the receptacles 550 are configured to accept
commonly used containers in the field of laboratory analysis, or
containers suitably configured for use with the holder herein. The
containers are typically stored separately from the holders to
facilitate sample handling, since solid reagents normally require
different storage conditions from liquid reagents. In particular
many solid reagents may be extremely moisture sensitive.
[0055] The snapped-in reagent tubes containing different reagents
may be of different colors, or color-coded for easy identification
by the user. For example they may be made of different color
material, such as tinted plastic, or may have some kind of
identifying tag on them, such as a color stripe or dot. They may
also have a label printed on the side, and/or may have an
identifier such as a barcode on a sealing layer on their respective
tops.
[0056] The containers 554 received by the receptacles 550 may
alternatively be an integrated part of the holder and may be the
same type of container as the waste chamber and/or the reagent
tube(s), or may be different therefrom.
[0057] In one embodiment, the containers 554 containing lyophilized
reagents, disposed in the receptacles 550, are 0.3 ml tubes that
have been further configured to have a star-shaped pattern on their
respective bottom interior surfaces. This is so that when a fluid
has been added to the lyophilized reagents (which are dry in the
initial package), a pipette tip can be bottomed out in the tube and
still be able to withdraw almost the entire fluid from the tube.
The design of the star-pattern is further described elsewhere in
U.S. patent application Ser. No. 12/______, filed on even date
herewith, and entitled "Reagent Tube", in the name of Handique et
al., which application is incorporated herein by reference.
[0058] The embodiment of a reagent holder 804 is shown configured
with a waste chamber 560, having an inlet aperture 562 in the upper
side of the connecting member. Waste chamber 560 is optional and,
in embodiments where it is present, is configured to receive spent
liquid reagents. In other embodiments, where it is not present,
spent liquid reagents can be transferred to and disposed of at a
location outside of the holder, such as, for example, a sample tube
that contained the original sample whose contents are being
analyzed. Waste chamber 560 is shown as part of an assembly
comprising additionally two or more reagent tubes 540. It would be
understood that such an arrangement is done for convenience, e.g.,
of manufacture; other locations of the waste chamber are possible,
as are embodiments in which the waste chamber is adjacent a reagent
tube, but not connected to it other than via the connecting
member.
[0059] The embodiment of a reagent holder 804 is shown having a
pipette sheath 570. This is an optional component of the holders
described herein. It may be permanently or removably affixed to
connecting member 510, or may be formed, e.g., moulded, as a part
of a single piece assembly for the holder. Pipette sheath 570 is
typically configured to surround the at least one socket and a tip
and lower portion of a pipette tip when the pipette tip is
stationed in the at least one socket. In some embodiments, the at
least one socket comprises four sockets. In some embodiments the at
least one socket comprises two, three, five, or six sockets.
[0060] Pipette sheath 570 typically is configured to have a bottom
576 and a walled portion 578 disposed between the bottom and the
connecting member. Pipette sheath 570 may additionally and
optionally have one or more cut-out portions 572 in the wall 578,
or in the bottom 576. Such cutouts provide ventilation for the
pipette tips and also reduce the total amount of material used in
manufacture of the holder, and may have various numbers, positions,
shapes and sizes, as further described in U.S. patent application
Ser. No. 12/______, filed by ExpressMail on Jul. 14, 2008, entitled
"Reagent Tube, Reagent Holder, and Kits Containing Same", in the
name of Wilson, et al., and incorporated herein by reference.
[0061] In embodiments having a pipette sheath, a purpose of the
sheath is to catch drips from used pipette tips, and thereby to
prevent cross-sample contamination, from use of one holder to
another in a similar location, and/or to any supporting rack in
which the holder is situated. Typically, then, the bottom 576 is
solid and bowl-shaped (concave) so that drips are retained within
it. An embodiment having no pipette sheath, could utilize, e.g., a
drip tray or a drainage outlet, suitably placed beneath pipette
tips located in the one or more sockets, for the same purpose and
located under or in the bottom of the rack, as described herein. In
addition to catching drips, the pipette tip sheath prevents or
inhibits the tips of other reagent holders--such as those that are
situated adjacent to the one in question in a rack as further
described herein--from touching each other when the tips are picked
up and/or dropped off before or after some liquid processing step.
Contact between tips in adjacent holders is generally not intended
by, for example, an automated dispensing head that controls sample
processing on holders in parallel, but the pipette tips being long
can easily touch a tip in a nearby holder if the angle when
dropping off of the tip deviates slightly from vertical.
[0062] Process tube 520 can also be a snap-in tube, rather than
being part of an integrated piece. Process tube 520 is typically
used for various mixing and reacting processes that occur during
sample preparation. For example, cell lysis can occur in process
tube 520, as can extraction of nucleic acids, such as DNA or RNA of
a patient, and DNA or RNA of a pathogen. Process tube 520 is then
advantageously positioned in a location that minimizes, overall,
pipette head moving operations involved with transferring liquids
to process tube 520. Process tube 520 is also located in the holder
in such a position that, when the holder is inserted in a rack as
further described herein, the process tube is exposed and
accessible to a heater and separator, as further described
herein.
[0063] Some of the reagents contained in the holder are provided as
liquids, and others may be provided as solids. In some embodiments,
a different type of container or tube is used to store liquids from
those that store the solids.
[0064] Reagent tubes 540 are typically configured to hold liquid
reagents, one per tube. For example, in reagent holder embodiment
804, three reagent tubes are shown, containing respectively wash
buffer, release buffer, and neutralization buffer, each of which is
used in a sample preparation protocol.
[0065] Reagent tubes 540 that hold liquids or liquid reagents can
be sealed with a laminate structure 598 to facilitate long term
storage. The laminate structure typically has a heat seal layer, a
plastic layer such as a layer of polypropylene, and a layer of
metal such as aluminum foil, wherein the heat seal layer is
adjacent the one or more reagent tubes. The additional plastic film
that is used in a laminate for receptacles that contain liquid
reagents is typically to prevent liquid from contacting the
aluminum. Various embodiments of a laminate structure are described
in U.S. patent application Ser. No. 12/______, filed by ExpressMail
on Jul. 14, 2008, entitled "Reagent Tube, Reagent Holder, and Kits
Containing Same", in the name of Wilson, et al., and incorporated
herein by reference. In some embodiments, a raised flat portion 599
facilitates application and removal of laminate 598. Raised surface
599, on the upper side of the connecting member, and surrounding
the inlet apertures to the reagent tubes and, optionally, the waste
chamber, is an optional feature of the holder.
[0066] The containers that hold solid, such as lyophilized
reagents, can be sealed across their tops by a metal foil, such as
an aluminum foil, with no plastic lining layer, as further
described herein.
[0067] The reagent holder 804 has a connecting member that is
configured so that the at least one socket, the one or more
receptacles, and the respective apertures of the process tube, and
the two or more reagent tubes, are all arranged linearly with
respect to one another (i.e., their midpoints lie on the same
axis). However, the holders herein are not limited to particular
configurations of receptacles, process tube, sockets, reagent
tubes, and waste chamber if present. For example, a holder may be
made shorter, if some apertures are staggered with respect to one
another and occupy `off-axis` positions. The various receptacles,
etc., also do not need to occupy positions with respect to one
another that are the same as those shown in FIGS. 7A and 7B. Thus,
in FIGS. 7A, and 7B, the process tube is on one end of the
connecting member, and the pipette sheath is at the other end,
adjacent to, in an interior position, a waste chamber and two or
more reagent tubes. Still other dispositions are possible, such as
mounting the process tube on one end of the holder, mounting the
process tube adjacent the pipette tips and pipette tip sheath (as
further described herein), and mounting the waste tube adjacent the
process tube. It would be understood that alternative
configurations of the various parts of the holder give rise only to
variations of form and can be accommodated within other variations
of the apparatus as described, including but not limited to
alternative instruction sets for a liquid dispensing pipette head,
heater assembly, and magnetic separator, as further described
herein. Each such configuration of the reagent holder can be
accommodated by a corresponding variation in form of the rack
described herein that receives one or more such holders.
[0068] The materials of the various tubes and chambers may be
configured to have at least an interior surface smoothness and
surface coating to reduce binding of DNA and other macromolecules
thereto. Binding of DNA is unwanted because of the reduced
sensitivity that is likely to result in subsequent detection and
analysis of the DNA that is not trapped on the surface of the
holder.
[0069] The process tube also may have a low binding surface, and
allows magnetic beads to slide up and down the inside wall easily
without sticking to it. Moreover, it has a hydrophobic surface
coating enabling low stiction of fluid and hence low binding of
nucleic acids and other molecules.
[0070] In some embodiments, the holder comprises a registration
member such as a mechanical key. Typically such a key is part of
the connecting member 510. A mechanical key ensures that the holder
is accepted by a complementary member in, for example, a supporting
rack as described herein or a receiving bay of an apparatus that
controls pipetting operations on reagents in the holder. A
mechanical key is normally a particular-shaped cut-out that matches
a corresponding cutout or protrusion in a rack or receiving
apparatus. Thus, embodiment 804 has a mechanical key 592 that
comprises a pair of rectangular-shaped cut-outs on one end of the
connecting member. This feature as shown additionally provides for
a tab by which a user may gain a suitable purchase when inserting
and removing the holder into a rack or another apparatus.
Embodiment 804 also has a mechanical key 590 at the other end of
connecting member 510. Key 590 is an angled cutout that eases
insertion of the holder into a rack, as well as ensures a good
registration therein when abutting a complementary angled cut out
in a recessed area configured to receive the holder. Other
variations of a mechanical key are, of course, consistent with the
description herein: for example, curved cutouts, or various
combinations of notches or protrusions all would facilitate secure
registration of the holder.
[0071] In some embodiments, not shown in FIGS. 7A, 7B, the holder
further comprises an identifier affixed to the connecting member.
The identifier may be a label, such as a writable label, a
bar-code, a 2-dimensional bar-code, or an RFID tag. The identifier
can be, e.g., for the purpose of revealing quickly what combination
of reagents is present in the holder and, thus, for what type of
sample preparation protocol it is intended. The identifier may also
indicate the batch from which the holder was made, for quality
control or record-keeping purposes. The identifier may also permit
a user to match a particular holder with a particular sample.
[0072] It should also be considered consistent with the description
herein that a holder additionally can be configured to accept a
sample, such as in a sample tube. Thus, in embodiments described
elsewhere herein, a rack accepts a number of sample tubes and a
number of corresponding holders in such a manner that the sample
tubes and holders can be separately and independently loaded from
one another. Nevertheless, in other embodiments, a holder can be
configured to also accept a sample, for example in a sample tube.
And thus, a complementary rack is configured to accept a number of
holders, wherein each holder has a sample as well as reagents and
other items. In such an embodiment, the holder is configured so
that the sample is accessible to a sample identification
verifier.
[0073] A reagent holder for use with a rack as described herein is
typically made of a plastic such as polypropylene. The plastic is
such that it has some flexibility to facilitate placement into a
rack, as further described herein. The plastic is typically
sufficiently rigid, however, so that the holder will not
significantly sag or flex under its own weight and will not easily
deform during routine handling and transport, and thus will not
permit reagents to leak out from it.
[0074] The holder is typically such that the connecting member,
process tube, the two or more reagent tubes, and the waste chamber
(if present) are made from a single piece, made from a material
such as polypropylene.
[0075] The reagent holder for use herein is also typically such
that at least the process tube, and the two or more reagent tubes
are translucent.
[0076] The reagent holders can be disposable, such as intended for
a single use, following which it is discarded.
Liquid Dispenser
[0077] Additionally, the holders herein are configured, when
received by a rack as described elsewhere herein, to accept pipette
operations both performed manually by an operator, and by an
automated pipette head. An exemplary automated pipette head is
described in U.S. provisional application Ser. No. 60/959,437,
filed Jul. 13, 2008, and in U.S. patent application Ser. No.
12/173,023, filed Jul. 14, 2008, entitled "Integrated Apparatus for
Performing Nucleic Acid Extraction and Diagnostic Testing on
Multiple Biological Samples", in the name of Williams, et al., both
of which are incorporated herein by reference in their entirety.
Typical features of an automated pipette head suitable for
operating with holders as described herein include at least: an
ability to pick up pipette tips from the one or more sockets, and
to return pipette tips to such sockets after use; to strip and
discard a pipette tip from a pipette head after use or upon
encountering an error; move a pipette tip with precision from one
location of a given holder to another so that, for example, liquid
reagents can be located and added to solid reagents to make up
solutions, and various liquid reagents can be mixed with one
another during a sample preparation protocol. Furthermore, it is
desirable that such an automated pipette device can operate on
several, such as 2, 3, 4, or 6, holders simultaneously when
received by a rack, and thereby perform certain operations in
parallel. Thus the pipette head should move in three degrees of
freedom.
Heater Assembly & Magnetic Separator
[0078] The racks as described herein are configured such that
reagent holders are positioned so that the process tube is heated
by a dedicated heating unit 977, for example situated in an
apparatus for carrying out sample preparation on multiple samples
in parallel, as shown in FIG. 2. Typically such a heater assembly
comprises one or more independently controllable heater units 1010,
each of which comprises a heat block configured to heat a process
tube in a reagent holder situated in the rack, as further described
herein. In one embodiment, a heat element is a power resistor. The
right hand panel of FIG. 2 shows how holders loaded in a rack can
be positioned in close proximity to a dedicated heating unit. The
heating unit is configured to heat the process tube in each of one
or more reagent holders positioned in the rack, without unduly
heating other portions of the rack, or other containers associated
with the reagent holders.
[0079] Yet additionally, the holders herein are configured so that
the process tube is in close enough proximity to a magnetic
assembly that separation of magnetic particles from reagents in
solution in the process tube can be accomplished. An exemplary
magnetic separator is configured to move one or more magnets
relative to the one or more process tubes. Typically, the magnet is
mounted in such a way that it can be moved in proximity to the
process tubes, either in an automated fashion such as under control
of a processor, or manually. The magnet can be made of neodymium
(e.g., from K &J Magnetics, Inc.) and can have a magnetic
strength of 5,000-15,000 Gauss (Brmax). The poles of the magnets
can be arranged such that one pole faces the heat blocks and the
other faces away from the heat blocks.
[0080] Advantageously, the heater assembly and magnetic separator
operate together to permit successive heating and separation
operations to be performed on liquid materials in the one or more
process tubes without transporting either the liquid materials or
the process tubes to different locations to perform either heating
or separation. An exemplary heater assembly and magnetic separator
are further described in U.S. provisional Patent Application Ser.
No. 60/959,437, filed Jul. 13, 2008, and U.S. patent application
Ser. Nos. 12/173,023, filed Jul. 14, 2008, entitled "Integrated
Apparatus for Performing Nucleic Acid Extraction and Diagnostic
Testing on Multiple Biological Samples", in the name of Williams,
et al. and "Ser. No. 12/______, entitled "Integrated Heater and
Magnetic Separator", in the name of Handique, filed on even date
herewith, all of which are incorporated herein by reference in
their entirety.
Apparatus Overview
[0081] A schematic overview of an apparatus 981 for carrying out
automated sample preparation on multiple samples in parallel,
according to steps exemplified elsewhere herein, is shown in FIG.
8. The geometric arrangement of the components of system 981 is
schematic and not intended to be limiting. The apparatus may
additionally comprise (not shown in FIG. 8) a microfluidic
cartridge, in a receiving bay, and configured to carry out a
diagnostic test on the sample, such as by detecting presence of an
amplified polynucleotide in the cartridge. Such additional features
are also described in U.S. patent application Ser. No. 12/173,023,
filed on Jul. 14, 2008 (and entitled "Integrated Apparatus for
Performing Nucleic Acid Extraction and Diagnostic Testing on
Multiple Biological Samples", in the name of Williams, et al.).
[0082] A processor 980, such as a microprocessor, is configured to
control functions of various components of the system as shown, and
is thereby in communication with each such component requiring
control. It is to be understood that many such control functions
can optionally be carried out manually, and not under control of
the processor. Furthermore, the order in which the various
functions are described, in the following, is not limiting upon the
order in which the processor executes instructions when the
apparatus is operating. Thus, processor 980 can be configured to
receive data about a sample to be analyzed, e.g., from a sample
reader 990, which may be a barcode reader, an optical character
reader, or an RFID scanner (radio frequency tag reader).
[0083] Processor 980 can be configured to accept user instructions
from an input device 984, where such instructions may include
instructions to start analyzing the sample, and choices of
operating conditions. Processor 980 can be also configured to
communicate with a display 982, so that, for example, information
about an analysis is transmitted to the display and thereby
communicated to a user of the system. Such information includes but
is not limited to: the current status of the apparatus; progress of
PCR thermocycling; and a warning message in case of malfunction of
either system or cartridge. Additionally, processor 980 may
transmit one or more questions to be displayed on display 982 that
prompt a user to provide input in response thereto. Thus, in
certain embodiments, input 984 and display 982 are integrated with
one another. Processor 980 can be optionally further configured to
transmit results of an analysis to an output device 986 such as a
printer, a visual display, a display that utilizes a holographic
projection, or a speaker, or a combination thereof. Processor 980
can be still further optionally connected via a communication
interface such as a network interface to a computer network
988.
[0084] Processor 980 can be further configured to control various
aspects of sample preparation and diagnosis, as follows in
overview. In FIG. 11, the apparatus 981 is configured to operate in
conjunction with a complementary rack 800. Apparatus 981 may be
capable of receiving multiple racks, such as 1, 2, 3, 4, or 6
racks.
[0085] A rack 800 is itself configured, as further described
herein, to receive a number of biological samples 996 in a form
suitable for work-up and diagnostic analysis, and a number of
holders 804--as further described herein, such as in connection
with FIGS. 7A, 7B, that are equipped with various reagents, pipette
tips and receptacles. The rack is configured so that, during sample
work-up, samples are processed in the respective holders, the
processing including being subjected, individually, to heating and
cooling via heater assembly 977.
[0086] The heating functions of the heater assembly can be
controlled by the processor 980. Heater assembly 977 operates in
conjunction with a separator 978, such as a magnetic separator,
that also can be controlled by processor 980 to move into and out
of close proximity to one or more processing chambers associated
with the holders 804, wherein particles such as magnetic particles
are present.
[0087] Liquid dispenser 976, which similarly can be controlled by
processor 980, is configured to carry out various suck and dispense
operations on respective sample, fluids and reagents in the holders
804, to achieve extraction of nucleic acid from the samples. Liquid
dispenser 976 can carry out such operations on multiple holders
simultaneously. Sample reader 990 is configured to transmit
identifying indicia about the sample, and in some instances the
holder, to processor 980. In some embodiments a sample reader is
attached to the liquid dispenser and can thereby read indicia about
a sample above which the liquid dispenser is situated. In other
embodiments the sample reader is not attached to the liquid
dispenser and is independently movable, under control of the
processor. Liquid dispenser 976 is also configured to take aliquots
of fluid containing nucleic acid extracted from one or more samples
and direct them to storage area 974, which may be a cooler. Area
974 contains, for example, a PCR tube corresponding to each
sample.
[0088] Embodiments of the apparatus shown in outline in FIG. 8, as
with other exemplary embodiments described herein, are advantageous
because they do not require locations within the apparatus suitably
configured for storage of reagents. Therefore, the apparatus in
FIG. 8 is self-contained and operates in conjunction with holders
804, wherein the holders are pre-packaged with reagents, such as in
locations within it dedicated to reagent storage.
[0089] The apparatus of FIG. 8 may be configured to carry out
operation in a single location, such as a laboratory setting, or
may be portable so that they can accompany, e.g., a physician, or
other healthcare professional, who may visit patients at different
locations. The apparatus is typically provided with a power-cord so
that they can accept AC power from a mains supply or generator. The
apparatus may also be configured to operate by using one or more
batteries and therefore is also typically equipped with a battery
recharging system, and various warning devices that alert a user if
battery power is becoming too low to reliably initiate or complete
a diagnostic analysis.
[0090] The apparatus of FIG. 8 may further be configured, in other
embodiments, for multiplexed sample analysis and/or analysis of
multiple batches of samples, where, e.g., a single rack holds a
single batch of samples. Each component shown in FIG. 8 may
therefore be present as many times as there are batches of samples,
though the various components may be configured in a common
housing.
[0091] The apparatus herein can be configured to run on a
laboratory benchtop, or similar environment, and can test
approximately 45 samples per hour when run continuously throughout
a normal working day. Results from individual raw samples are
typically available in less than 1 hour.
EXAMPLES
Example 1
Reagent Holder
[0092] An exemplary reagent holder consistent with the description
herein, and suitable for use with a rack as described herein, has
the following dimensions and capacities:
[0093] 180 mm long X 22 mm wide X 100 mm tall;
[0094] Made from Polypropylene.
[0095] One snapped-in low binding 1.7 ml tube that functions as a
process tube.
[0096] 3 built-in tubes that function as receptacles for reagents,
as follows: [0097] One tube containing 200-1000 .mu.l of wash
buffer (0.1 mM Tris, pH 8). [0098] One tube containing 200-1000
.mu.l of release solution (40 mM NaOH). [0099] One tube containing
200-1000 .mu.l of neutralization solution (330 mM Tris, pH
8.0).
[0100] One built-in tube that functions as a waste chamber (will
hold .about.4 ml of liquid waste).
[0101] 3 receptacles to accept containers for solid reagents.
Snap-in 0.3 ml or 0.65 ml PCR tubes (which are typically stored
separately from the reagent holder) are placed in each of these
locations, and contain, respectively: [0102] lyophilized sample
preparation reagents (lysis enzyme mix and magnetic affinity
beads). [0103] First lyophilized PCR master mix, probes and primers
for a first target analyte detection. [0104] Second lyophilized PCR
master mix, probes and primers for a second target analyte
detection (only offered in select cases, such as detection of
Chlamydia and Gonorrhea from urine).
[0105] 4 pipette tips located in 4 respective sockets.
[0106] Pipette tip Sheath: The pipette tips have a sheath/drip tray
underneath to help capture any drip from the pipette tips after
being used, and also to prevent unwanted contamination of the
instrument.
[0107] A mechanical key, in conjunction with a spring, allows easy
insertion, removal, and positive location of strip in rack.
[0108] One or more labels: positioned upward facing to facilitate
ease of reading by eye and/or, e.g., a bar-code reader, the one or
more labels containing human and machine readable information
pertaining to the analysis to be performed.
[0109] It is to be understood that these dimensions are exemplary.
However, it is particularly desirable to ensure that a holder does
not exceed these dimensions so that a rack and an apparatus that
accommodates the reagent holder(s) does not become inconveniently
large, and can be suitably situated in a laboratory, e.g., on a
bench-top.
Example 2
Exemplary Chemistry Processes Performed by an Automated
Instrument
Sample Pre-Processing
[0110] For Urine Sample: Take 0.5 ml of urine and mix it with 0.5
ml of collection buffer. Filter the sample through a pre-filter
(containing two membranes of 10 micron and 3 micron pore size).
Place the sample tube in the position specified for the external
sample tube in a 12-holder rack.
[0111] For Plasma Sample: Take 0.5 ml of plasma and mix it with 0.5
ml of collection buffer. Place the sample tube in the position
specified for the external sample tube in the 12-holder rack.
[0112] For GBS swab samples: Take the swab sample and dip it in 1
ml of collection buffer. Place the sample tube in the position
specified for the external sample tube in the 12-holder rack.
[0113] The sample collection buffer contains 50 mM Tris pH 7, 1%
Triton X-100, 20 mM Citrate, 20 mM Borate, 100 mM EDTA, plus 1,000
copies of positive control DNA.
Loading the Instrument and Starting Sample Processing
[0114] The following steps may be performed to initiate an analysis
on samples in batch. [0115] 1. Load PCR tube containing PCR master
mix in one of the specified snap-in location of the reagent holder.
[0116] 2. Load PCR tube containing PCR probes and primers for the
target analyte under consideration in the specified location of the
reagent holder. [0117] 3. In case of two analyte test, load PCR
tube containing probes and primers for second analyte in the
specified location of the reagent holder. [0118] 4. Insert the
reagent holder in a 12-holder rack in the same lane as the sample
tube under consideration. [0119] 5. Prepare and insert reagent
holders for other samples in consideration. [0120] 6. Load the
12-holder rack in one of the locations in the instrument. [0121] 7.
Load a 12-sample cartridge in the cartridge tray loading position.
[0122] 8. Start operation.
Liquid Processing Steps
[0123] The following steps may be performed to carry out sample
preparation. [0124] 1. Using Pipette tip #1, the robot transfers
the clinical sample from the external sample tube to the process
tube of the reagent holder. [0125] 2. Using the same pipette tip,
the robot takes about 100 .mu.l of sample, mixes the lyophilized
enzyme and affinity beads, transfers the reagents to the process
tube. Mixing is performed in the process tube by 5 suck and
dispense operations. [0126] 3. The robot places pipette tip #1 at
its designated location in the reagent holder. [0127] 4. Heat the
process tube to 60.degree. C. and maintain it for 10 minutes.
[0128] 5. After 5 minute of lysis, the robot picks up pipette tip
#1 and mixes the contents by 3 suck and dispense operations. [0129]
6. The robot places pipette tip #1 at its designated location in
the reagent holder. [0130] 7. After 10 minutes of lysis, a magnet
is moved up the side of the process tube to a middle height of the
sample and held at that position for a minute to capture all the
magnetic beads against the wall the tube. [0131] 8. The magnet is
brought down slowly to slide the captured beads close to the bottom
(but not the bottom) of the tube. [0132] 9. Using pipette tip #2,
aspirate all the liquid and dump it into the waste tube. [0133] 10.
Aspirate a second time to remove as much liquid as possible from
the process tube. [0134] 11. Using the same pipette tip #2,
withdraw 100 .mu.l of wash buffer and dispense it in the process
tube. During this dispense, the magnet is moved downwards, away
from the process tube. [0135] 12. Perform 15 mix steps to
thoroughly mix the magnetic beads with the wash buffer. [0136] 13.
Wait for 30 seconds. [0137] 14. Move magnet up to capture the beads
to the side and hold for 15 seconds. [0138] 15. Using pipette tip
#2, aspirate wash buffer twice to remove as much liquid as possible
and dump it back in the wash tube. [0139] 16. Move magnet down away
from the process tube. [0140] 17. Place pipette tip #2 in its
specified location of the reagent holder. [0141] 18. Pick up a new
pipette tip (tip #3) and withdraw 8-10 .mu.l of release buffer and
dispense it over the beads in the process tube. [0142] 19. Wait for
1 minute and then perform 45 mixes. [0143] 20. Heat the release
solution to 85.degree. C. and maintain temperature for 5 minutes.
[0144] 21. Place pipette tip #3 in its specified location of the
reagent holder. [0145] 22. Bring magnet up the tube, capture all
the beads against the tube wall and move it up and away from the
bottom of the tube. [0146] 23. Pick up a new pipette tip (tip #4)
and withdraw all the release buffer from the process tube and then
withdraw 3-10 .mu.l of neutralization buffer, mix it in the pipette
tip and dispense it in the PCR tube. (In case of two analyte
detections, dispense half of the neutralized DNA solution into
first PCR tube and the rest of the solution in the second PCR tube.
[0147] 24. Using pipette tip #4, mix the neutralized DNA with the
lyophilized reagents by 4-5 suck and dispense operations and
withdraw the entire solution in the pipette tip. [0148] 25. Using
pipette tip #4, load 6 .mu.l of the final PCR solution in a lane of
the 12-up cartridge.
Real-Time PCR
[0149] After all the appropriate PCR lanes of the PCR cartridge are
loaded with final PCR solution, the tray containing the cartridge
moves it in the PCR Analyzer. The cartridge is pressed by an
optical detection read-head against the PCR heater. Heaters
activate valves to close either ends of the PCR reactor and
real-time thermocycling process starts. After completing
appropriate PCR cycles (.about.45 cycles), the analyzer decides
whether the sample has the target DNA based on the output
fluorescence data, and issues an indication of the same.
Example 3
Exemplary Rack
[0150] In an exemplary rack for use, in pairs, with a diagnostic
instrument, disposable reagent holders (e.g., oriented orthogonally
to the front of the instrument) can be contained in one of two,
12-lane racks. The 12 reagent holders in a given rack can register
and lock into the rack upon insertion by a user. The rack contains
an area for 12 sample lysis tubes and holds the tube bottoms
co-planar, thereby allowing the user to orient a bar code on each
tube to face the rear of the instrument.
[0151] Certain features, permit the racks to be inserted and
oriented in the instrument by a minimally trained user. Proper rack
placement can be confirmed by feedback to the software. In some
embodiments, the racks can be black and color fast (e.g., the color
may not appreciably degrade with use or washing with a 10% bleach
solution) and the rack material can be dimensionally stable to
within 0.1 mm over the operating temperature range of the system.
The rack can be designed with provisions to allow the rack to be
carried to and from the instrument and to minimize or eliminate the
likelihood that the tubes held by the rack will spill when placed
on a flat surface.
[0152] The foregoing description is intended to illustrate various
aspects of the present inventions. It is not intended that the
examples presented herein limit the scope of the present
inventions. The technology now being fully described, it will be
apparent to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the appended claims.
* * * * *