U.S. patent application number 15/715063 was filed with the patent office on 2018-08-16 for methods and systems for pure dye instrument normalization.
This patent application is currently assigned to Life Technologies Corporation. The applicant listed for this patent is Life Technologies Corporation. Invention is credited to Jeffrey Marks.
Application Number | 20180230511 15/715063 |
Document ID | / |
Family ID | 55447126 |
Filed Date | 2018-08-16 |
United States Patent
Application |
20180230511 |
Kind Code |
A1 |
Marks; Jeffrey |
August 16, 2018 |
METHODS AND SYSTEMS FOR PURE DYE INSTRUMENT NORMALIZATION
Abstract
The present teachings relate to a method and system for
normalizing spectra across multiple instruments. In an embodiment
of the present invention, the method comprises at least one
reference instrument and a test instrument. Each instrument
comprises at least one excitation filter and at least one emission
filter arranged in pairs. Each instrument further comprises a pure
dye plate comprising a plurality of wells. Each well contains a
plurality of dyes where each dye comprises a fluorescent component.
Fluorescent spectra are obtained from each instrument for each dye
across multiple filter combinations to contribute to a pure dye
matrix Mref for the reference instrument and pure dye matrix M for
the test instrument. The pure dye spectra can then be multiplied by
correction factors for each filter pair to result in corrected pure
dye spectra, then normalized and the multicomponenting data can be
extracted.
Inventors: |
Marks; Jeffrey; (Mountain
View, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Life Technologies Corporation |
Carlsbad |
CA |
US |
|
|
Assignee: |
Life Technologies
Corporation
Carlsbad
CA
|
Family ID: |
55447126 |
Appl. No.: |
15/715063 |
Filed: |
September 25, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15016713 |
Feb 5, 2016 |
9809849 |
|
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15715063 |
|
|
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62112964 |
Feb 6, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2021/6421 20130101;
G01N 2201/04 20130101; G01N 21/6452 20130101; G01N 21/6428
20130101; G01N 21/274 20130101; G01N 2201/068 20130101; G01N
2201/12746 20130101; C12Q 1/686 20130101 |
International
Class: |
C12Q 1/686 20180101
C12Q001/686; G01N 21/64 20060101 G01N021/64; G01N 21/27 20060101
G01N021/27 |
Claims
1. A method for normalizing laboratory instruments with pure dyes,
comprising: providing at least one reference instrument and a test
instrument, each instrument comprising at least one excitation
filter and at least one emission filter arranged in pairs;
providing a plurality of pure dyes, each dye comprising a
fluorescent component and contained in a pure dye plate comprising
a plurality of wells; generating fluorescent spectra from the
reference instrument and the test instrument for multiple pure dyes
across multiple filter combinations; creating a pure dye matrix,
Mref, for the reference instrument and a pure dye matrix, M, for
the test instrument; calculating correction factors for each the
adjustment factors filter pair and multiplying the correction
factors by the pure dye spectra; normalizing the corrected pure dye
spectra; generating multicomponent data.
2. The method of claim 1, wherein the fluorescent spectra from the
test and reference instruments are each normalized to a maximum of
1.
3. The method of claim 2, wherein each pure dye matrix comprises
normalized spectra averaged over multiple wells.
4. The method of claim 1, wherein dye matrix M is multiplied by a
set of adjustment factors and compared to dye matrix Mref.
5. The method of claim 4, wherein the adjustment factors are
iteratively modified until the difference between matrix M and
matrix Mref is minimized.
6. The method of claim 4, wherein the adjustment factors are
iteratively modified between 0 and 1.
7. The method of claim 1, wherein the correction factor for each
filter pair is the product of an emission filter factor and an
excitation filter factor.
8. The method of claim 1, wherein the corrected pure dye spectra
are normalized to a maximum of 1.
9. The method of claim 1, wherein the multicomponent data is the
product of the fluorescence data and the pseudo-inverse of dye
matrix M.
10. A system for normalizing laboratory instruments with pure dyes,
the system comprising: a pure dye reference matrix, Mref; a test
instrument, each comprising: a plurality of filter pairs; and at
least one pure dye plate. a computer system in communication with
the test instrument comprising: at least one processor; and at
least one computer-readable medium comprising instructions for pure
dye normalization executable by the processor.
11. The system of claim 10, wherein the filter pairs each comprise
an excitation filter and an emission filter.
12. The system of claim 10, wherein the pure dye plate comprises at
least one fluorescent pure dye contained in a sample plate
comprising a plurality of sample wells.
13. The system of claim 10, wherein the processor executes
instructions designed to generate a test spectra matrix M.
14. The system of claim 13, wherein matrix Mref and matrix M
comprise normalized and averaged spectra.
15. The system of claim 13, wherein the processor further executes
instructions designed to iteratively adjust matrix M until the
difference between matrix M and matrix Mref is minimized.
16. The system of claim 15, wherein the processor further executes
instructions designed to modify matrix M based on correction
factors for each filter pair.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S.
provisional application Ser. No. 62/112,964, filed Feb. 6, 2015,
which is incorporated herein by reference in its entirety.
BACKGROUND
[0002] Currently, genomic analysis, including that of the estimated
30,000 human genes is a major focus of basic and applied
biochemical and pharmaceutical research. Such analysis may aid in
developing diagnostics, medicines, and therapies for a wide variety
of disorders. However, the complexity of the human genome and the
interrelated functions of genes often make this task difficult. One
difficulty commonly faced is the inability of researchers to easily
compare results of experiments run on multiple instruments.
Physical variations in the parameters of components such as light
sources, optical elements and fluorescence detectors, for example,
can result in variation in the results of analyses on what may be
identical biological samples. There is, therefore, a continuing
need for methods and apparatus to aid in minimizing the variations
in the components. One such methodology is described in the present
teachings.
DRAWINGS
[0003] One skilled in the art will understand that the drawings,
described herein, are for illustration purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way.
[0004] FIG. 1 illustrates a computer system on which embodiments of
the present teachings can be implemented.
[0005] FIG. 2 illustrates a laboratory instrument upon which
embodiments of the present teachings can be implemented.
[0006] FIG. 3 illustrates actual and estimated normalization
factors according to embodiments of the present teachings.
[0007] FIG. 4A illustrates dye mixtures used in various embodiments
of the present teachings.
[0008] FIG. 4B illustrates pure dyes and main channel filter
combinations for various embodiments of the present teachings.
[0009] FIG. 5 illustrates percent deviation of dye mixtures before
normalization according to various embodiments of the present
teachings.
[0010] FIG. 6 illustrates percent deviation of dye mixtures after
normalization according to various embodiments of the present
teachings.
[0011] FIG. 7 illustrates a closer view of percent deviation of dye
mixtures after normalization according to various embodiments of
the present teachings.
[0012] FIG. 8 is a flow chart depicting a normalization process
according to various embodiments of the present teachings.
[0013] FIG. 9 illustrates the correlation between a test instrument
and a reference instrument before and after normalization according
to various embodiments of the present teachings.
SUMMARY OF THE INVENTION
[0014] The present teachings relate to a method and system for
normalizing spectra across multiple instruments. In an embodiment
of the present invention, the method comprises at least one
reference instrument and a test instrument. Each instrument
comprises at least one excitation filter and at least one emission
filter arranged in pairs. Each instrument further comprises a pure
dye plate comprising a plurality of wells. Each well contains a
plurality of dyes where each dye comprises a fluorescent component.
Fluorescent spectra are obtained from each instrument for each dye
across multiple filter combinations to contribute to a pure dye
matrix Mref for the reference instrument and pure dye matrix M for
the test instrument. The pure dye spectra can then be multiplied by
correction factors for each filter pair to result in corrected pure
dye spectra, then normalized and the multicomponenting data can be
extracted.
[0015] In another embodiment, the fluorescent spectra from the
reference instrument and the test instrument are first normalized
and then averaged over multiple wells to form the pure dye
matrices.
[0016] In another embodiment, dye matrix M is multiplied by a set
of adjustment factors that are iteratively modified to minimize the
difference between matrix M and matrix Mref.
[0017] In another embodiment, the adjustment factors are modified
between 0 and 1
[0018] In another embodiment, the correction factor is the product
of the emission filter factor and the excitation filter factor.
[0019] In another embodiment, the corrected pure dye spectra are
normalized to a value of one.
[0020] In another embodiment, the multicomponent data is derived
from the product of the fluorescence data and the pseudo-inverse
dye matrix M.
[0021] According to various embodiments, a system for normalizing
laboratory instruments with pure dyes is presented. The system can
comprise a pure dye reference matrix Mref. The system can further
comprise a test instrument. The test instrument can comprise a
plurality of filter pairs and at least one pure dye plate. The
system can further comprise a computer system in communication with
the test instrument and comprising at least one processor and at
least one computer-readable medium comprising instructions for pure
dye normalization executable by the processor.
[0022] In another embodiment, the filter pairs comprise an
excitation filter and an emission filter.
[0023] In another embodiment, the pure dye plate comprises at least
one fluorescent pure dye contained in a sample plate comprising a
plurality of sample wells.
[0024] In another embodiment, the processor executes instructions
designed to generate a test spectra matrix M.
[0025] In another embodiment, matrix Mref and matrix M comprise
normalized and averaged spectra.
[0026] In another embodiment, the processor further executes
instructions designed to iteratively adjust matrix M until the
difference between matrix M and matrix Mref is minimized.
[0027] In another embodiment, the processor further executes
instructions designed to modify matrix M based on correction
factors for each filter pair.
DESCRIPTION OF VARIOUS EMBODIMENTS
[0028] The following description of various embodiments is merely
exemplary in nature and is in no way intended to limit the present
teachings, applications or uses. Although the present teachings
will be discussed in some embodiments as relating to polynucleotide
amplification, such as polymerase chain reaction (PCR), such
discussion should not be regarded as limiting the present teaching
to only those applications.
[0029] FIG. 1 is a block diagram that illustrates a computer system
100 upon which embodiments of the present teachings may be
implemented. Computer system 100 includes a bus 102 or other
communication mechanism for communicating information, and a
processor 104 coupled with bus 102 for processing information.
Computer system 100 also includes a memory 106, which can be a
random access memory (RAM) or other dynamic storage device, coupled
to bus 102, and instructions to be executed by processor 104.
Memory 106 also may be used for storing temporary variables or
other intermediate information during execution of instructions,
corresponding to the methods and present teachings, to be executed
by processor 104. Computer system 100 further includes a read only
memory (ROM) 108 or other static storage device coupled to bus 102
for storing static information and instructions for processor 104.
A storage device 110, such as, for example, but not limited to a
solid-state disk, a magnetic disk or optical disk, is provided and
coupled to bus 102 for storing information and instructions.
[0030] Computer system 100 may be coupled via bus 102 to a display
112, such as, for example, but not limited to a cathode ray tube
(CRT) or liquid crystal display (LCD), for displaying information
to a computer user. An input device 114, including alphanumeric and
other keys, is coupled to bus 102 for communicating information and
command selections to processor 104. Another type of user input
device is cursor control 116, such as, for example, but not limited
to a mouse, a trackball or cursor direction keys for communicating
direction information and command selections to processor 104 and
for controlling cursor movement on display 112. This input device
typically has two degrees of freedom in two axes, a first axis
(e.g., x) and a second axis (e.g., y), that allows the device to
specify positions in a plane.
[0031] Consistent with certain embodiments of the present
teachings, setup and calibration of laboratory instruments can be
performed by computer system 100 in response to processor 104
executing one or more sequences of one or more instructions
contained in memory 106. Such instructions may be read into memory
106 from another computer-readable medium, such as, for example
storage device 110. Execution of the sequences of instructions
contained in memory 106 causes processor 104 to perform the process
states described herein. Alternatively hard-wired circuitry may be
used in place of, or in combination with, software instructions to
implement the present teachings. Thus, implementations of the
present teachings are not limited to any specific combination of
hardware circuitry and software.
[0032] The term "computer-readable medium" as used herein refers to
any media that participates in providing instructions to processor
104 for execution. Such a medium may take many forms, including but
not limited to, nonvolatile media, volatile media, and transmission
media. Nonvolatile media can include but not be limited to, for
example, optical or magnetic disks, such as storage device 110.
Volatile media can include but not be limited to dynamic memory,
such as memory 106. Transmission media can include but not be
limited to coaxial cables, copper wire, and fiber optics, including
the wires that comprise bus 102. Transmission media can also take
the form of acoustic or light waves, such as those generated during
radio-wave and infrared data communications.
[0033] Common forms of computer-readable media can include, for
example, but not be limited to a floppy disk, flexible disk, hard
disk, magnetic tape, or any other magnetic medium, a CDROM, any
other optical medium, punch cards, paper tape, any other physical
medium with patterns of holes, a RAM, PROM, EPROM, FLASH-EPROM, USB
drive, jump drive or any other memory chip or cartridge, a carrier
wave, or any other medium from which a computer can read.
[0034] Various forms of computer readable media may be involved in
carrying one or more sequences of one or more instructions to
processor 104 for execution. For example, the instructions may
initially be carried on magnetic disk of a remote computer. The
remote computer can load the instructions into its dynamic memory
and send the instructions over, for example, a telephone line using
a modem or wireless network. A modem local to computer system 100
can receive the data on the telephone line and use an infra-red
transmitter to convert the data to an infra-red signal. An
infra-red detector coupled to bus 102 can receive the data carried
in the infra-red signal and place the data on bus 102. Bus 102
carries the data to memory 106, from which processor 104 retrieves
and executes the instructions. The instructions received by memory
106 may optionally be stored on storage device 110 either before or
after execution by processor 104.
[0035] The present teachings are described with reference to
Real-Time Polymerase Chain Reaction (RT-PCR) instruments. In
particular, an embodiment of the present teachings is implemented
for RT-PCR instruments employing optical imaging of well plates.
Such instruments can be capable of simultaneously or sequentially
measuring signals from a plurality of samples or spots for
analytical purposes and often require calibration, including but
not limited to processes involving: identifying ROI (Regions of
Interest), determining background signal, uniformity and pure dye
spectral calibration for multicomponent analysis. Calibration may
also involve a RT-PCR verification reaction using a known sample
plate with an expected outcome. One skilled in the art will
appreciate that while the present teachings have been described
with examples pertaining to RT-PCR instruments, their principles
are widely applicable to other forms of laboratory instrumentation
that may require calibration and verification in order to ensure
accuracy and/or optimality of results.
[0036] The present teachings can be applied to RT-PCR instrument
systems. Such RT-PCR instruments are well known to one skilled in
the art. For example the present teachings can be applied to
instruments such as, for example, but not limited to the Applied
Biosystems Sequence Detection Systems 7500/7900/ViiA7 and Quant
Studio systems, the Roche Applied Science LightCycler.RTM. 2.0 PCR
amplification and detection system, the Bio-Rad MyiQ Single-Color
Real-Time PCR Detection System, or the Stratagene Mx3000P.TM.
Real-Time PCR System. Such instruments generally use some form of
imaging system. While the present teachings are discussed relative
to a CCD (charge-coupled detector) imaging system, the present
teachings can be easily adapted to any form of imaging system.
[0037] In a system with a CCD imaging system, a CCD camera images a
sample plate (typically a 96-well plate, although plates with other
numbers of wells can be used or sample blocks containing individual
tubes can also be used) at various selected dye fluorescent
emission wavelengths during a PCR run. In such instruments, the
wells are generally illuminated by an excitation light at
wavelengths appropriate to each dye. In order to use the RT-PCR
system to accurately monitor PCR amplification using the well
emission intensities, the system must be calibrated for each dye
emission.
[0038] FIG. 2 is a schematic illustration of a system used for
fluorescent signal detection in accordance with implementations of
the present invention. Detection system 200 is one example of a
spectral detection system which can be used for RT-PCR data
collection and processing in conjunction with aspects of the
present invention. As illustrated, detection system 200 includes an
excitation light source 202, at least one filter turret depicted by
turret 204, a detector 208, a microwell tray 210 and well optics
212. Turret 204 can comprise multiple excitation filters or
multiple emission filters or multiple excitation and emission
filters paired for a specific dye. As illustrated, turret 204
includes filter cubes 206. A first filter cube 206A can include an
excitation filter 214A, a beam splitter 216A and an emission filter
218A corresponding to one spectral species selected from a set of
spectrally distinguishable species to be detected. A second filter
cube 206B can include an excitation filter 214B, a beam splitter
216B and an emission filter 218B corresponding to a different
spectral species selected from the set of spectrally
distinguishable species to be detected.
[0039] Excitation light source 202 can be, for example, but not
limited to a laser, broad spectrum light source, an LED or other
type of excitation source capable of emitting a spectrum that
interacts with spectral species to be detected by system 200. In
this illustrated example, light source 202 emits a broad spectrum
of light filtered by either excitation filter 214A or excitation
filter 214B that passes through beam splitter 216A or beam splitter
216B and onto microwell tray 210 containing one or more spectral
species.
[0040] Light emitted from light source 202 can be filtered through
excitation filter 214A, excitation filter 214B or other filters
that correspond closely to the one or more spectral species. The
present teachings can be used with a plurality of spectrally
distinguishable dyes such as, for example, but not limited to one
or more of FAM, SYBR Green, VIC, JOE, TAMRA, NED CY-3, Texas Red,
CY-5, Mustang Purple, ROX (passive reference) or any other
fluorochromes that emit a signal capable of being detected. The
target spectral species for the selected excitation filter provides
the largest signal response while other spectral species with lower
signal strength in the band-pass region of the filter contribute
less signal response. Because the multiple fluorochromes may have
this overlapping excitation and emission spectra, it is useful to
apply a pure-dye matrix to correct for the small amount of
"cross-talk" (signal from one dye detected with more than one
filter set). This process is often referred to as
multicomponenting.
[0041] In RT-PCR, amplification curves are often determined by
normalizing the signal of a reporter dye to a passive reference dye
in the same solution. Examples of reporter dyes can include, but
not be limited to FAM, SYBR Green, VIC, JOE, TAMRA, NED CY-3, Texas
Red, CY-5. An example of a passive reference can be, for example,
but not limited to ROX. This normalization can be reported as
normalized fluorescence values labeled as "Rn". Passive reference
normalization enables consistent Rn values even if the overall
signal level is affected by liquid volume, or overall illumination
intensity. Passive reference normalization, however, cannot work
properly if the ratio in signal between the reporter dye and
reference dye varies, such as from instrument-to-instrument
differences in the spectrum of the illumination. In order to adjust
for these differences, normalization solutions can be manufactured
to normalize the ratio of reporter to passive reference. An example
of such a normalization solution can be a 50:50 mixture of FAM and
ROX, which can be referred to as a "FAM/ROX" normalization
solution.
[0042] This current method of instrument normalization, including
reading fluorescence from the dye mixture to get a "normalization
factor" to adjust Rn values requires additional expense. Typically,
it can require the manufacture of normalization solutions and
normalization plates, and additional time to run the additional
calibrations. Further, this method only works for the dye mixtures
you are calibrating with a standard paired filter set. A paired
filter set can be a combination of an excitation filter and an
emission filter. One skilled in the art will understand that the
inclusion of an additional dye would require a different
normalization solution and calibration process.
[0043] Manufacturing processes for producing the normalization
solutions also contribute to variations in the response of the
dyes. It has been found that it can be difficult to control dye
concentrations due to the lack of an absolute fluorescence
standard. In order to minimize these errors and variations it can
be advantageous to target the dye ratio of the solution to within
+/-15% of the desired mix, or within +/-10% of the desired mix from
the manufacturing process. The manufacturing process is typically
not controlled well enough to simply mix a 50:50 mixture of the
dyes and meet those specifications, so an additional step in the
process is necessary to adjust the dye mixture with a
fluorimeter.
[0044] Acceptable percent variations disclosed above have been
determined by studying the relationship between variation in dye
mixture and Cts. A Ct is a common abbreviation for a "threshold
cycle". RT-PCR, also known as Quantitative PCR or qPCR, can provide
a method for determining the amount of a target sequence or a gene
that is present in a sample. During PCR a biological sample can be,
for example, subjected to a series of 35 to 40 temperature cycles.
A cycle can have multiple temperatures. For each temperature cycle
the amount of target sequence can theoretically double and is
dependent on a number of factors not presented here. Since the
target sequence contains a fluorescent dye, as the amount of target
sequence increases i.e. amplified over the 35 to 40 temperature
cycles the sample solution fluoresces brighter and brighter at the
completion of each thermal cycle. The amount of fluorescence
required to be measured by a fluorescence detector is frequently
referred to as a "threshold", and the cycle number at which the
fluorescence is detected is referred to as the "threshold cycle" or
Ct. Therefore by knowing how efficient the amplification is and the
Ct, the amount of target sequence in the original sample can be
determined.
[0045] The tolerated percent variation described above can also be
related to the standard deviation of Ct shifts in the instrument.
It has been determined that a +/-15% variation in dye mixture can
result in a standard deviation of 0.2 Cts which can be 2 standard
deviations.
[0046] As presented above, the ability to reliably compare
experimental results from multiple instruments is desirable and
instrument-to-instrument variability is frequently an issue. This
variability can result from two sources; variability of components
within the instruments such as, for example, but not limited to
lamps and filters as well as variability over time such as, for
example lamp and filter aging. It would be advantageous to
implement a process through which experimental results from
multiple instruments can be reliably, easily and inexpensively
compared. The teachings found herein disclose such a process.
[0047] The amount of fluorescent signal of a sample in an optical
system can be dependent on several factors. Some of the factors can
include, but not be limited to, the wavelength of the fluorescence
light, the detector efficiency at that wavelength of fluorescence
light, the efficiency of the emission filter, the efficiency of the
excitation filter and the efficiency of the dye. The present
teachings suggest that instrument-to-instrument variability can be
minimized if the physical optical elements of the instruments could
be normalized.
[0048] In one embodiment the normalization factors can be derived
from pure dye spectra rather than from dye mixtures. Pure dyes can
be easier to manufacture than dye mixtures, because the
concentrations do not have to be exact, and there is only one
fluorescent component. This concept was tested by normalizing two
filter sets in an instrument using ten pure dyes and comparing the
results to the normalization obtained from using dye mixtures. The
normalization was implemented by determining a correction factor
for each excitation filter and emission filter. The resulting
correction factors can be used to normalize any combination of
dyes, even from different instruments. FIG. 3 shows the results of
such a comparison. The estimated normalization factors for the pure
dyes are shown in red and the measured normalization factors from
the mixed dye plates are shown in blue. One skilled in the art can
see that the difference between the two sets of data are within the
desired +/-15% variation presented previously.
[0049] In another embodiment, the normalization taught above was
applied to multiple instruments of various types. Eight dye mixture
solutions and ten pure dye solutions were created. Each solution
was pipetted into eight wells of three 96 well plates. Potential
spatial crosstalk was minimized by pipetting into every other well.
The dye mixtures used are shown in FIG. 4A and the pure dyes used
are shown in FIG. 4B. In addition, the instruments used included
six sets of filters. FIG. 4B further identifies the filter pairs
for the main optical channel for each pure dye. The excitation
filter is depicted with an "X" and the emission filter is depicted
with an "M".
[0050] In an effort to quantify the effectiveness of the
normalization process, the dye ratios were measured before and
after normalization. FIG. 5 shows the percent deviation of dye
mixtures from the average ratio for 17 tested instruments. The
instruments are labeled on the X-axis and the percent deviation is
on the Y-axis. One skilled in the art will notice that the
deviation across the instruments is frequently greater than the
desired +/-15% previously discussed. This data, therefore, shows a
need for an improved normalization process such as the current
teachings.
[0051] The current teachings were applied to all 17 instruments.
The normalization method determines a correction factor for each
individual filter rather than for each dye ratio. Because the
instruments provided 6 excitation and 6 emission filters, 12
factors were determined. The process is shown in FIG. 8 and
flowchart 800. In step 805, calibration spectra were generated for
multiple dyes across multiple filter combinations. For the
instruments being normalized, there were 10 pure dyes and 21 filter
combinations. In step 810, the spectra were normalized so the
maximum signal was 1. In step 815 the dye spectra are averaged
across multiple wells. This averaging will result in producing one
spectrum per dye. Collectively, the dye spectra can be referred to
as a dye matrix "M" containing dye and filter combinations. At this
point, a reference instrument is identified. The reference
instrument could be an instrument or group of instruments that the
test instruments will be normalized to. The same set of dye spectra
used in the test instrument can be obtained from the reference
instrument(s). In some embodiments the reference can be a group of
instruments. In such an embodiment the spectra for each dye can be
averaged across the group. This step is represented in flowchart
800 at step 820. As an example, the reference spectra can be
referred to as matrix "Mref".
[0052] In step 825 each of the 12 filters has an adjustment factor
initially set to 1. It can be desirable to multiply the adjustment
factors times matrix "M" while iteratively modifying the adjustment
factors between 0 and 1 and preferably between 0.04 and 1 until the
difference between matrix "M" and matrix "Mref" is minimized as
shown in step 830. In step 835, correction factors for each filter
pair are calculated. The correction factor for each filter pair is
the product of the emission filter factor times the excitation
filter factor. The main channel filter pairs are shown in FIG. 4B.
Once the correction factors for each filter pair has been
determined, each filter pair factor can then be multiplied by the
fluorescence data for the test instrument as well as for the pure
dye spectra. The corrected pure dye spectra can then be
renormalized to a maximum value of 1 as shown in step 845. The
final step in the process at step 850 is to generate multicomponent
data. One skilled in the art will understand the multicomponenting
procedure to be the product of the fluorescence data and the
pseudo-inverse of the dye matrix. The multicomponent values are
already normalized so it will not be necessary to make dye specific
corrections since the data has been normalized at the filter
level.
[0053] At the completion of normalization the percent deviation of
dye mixtures from the average ratio were calculated across all 17
instruments. The results are shown in FIG. 6. These results are
significantly improved as compared to the data before normalization
as shown in FIG. 5. A closer view of the normalized data from FIG.
6 is shown in FIG. 7, where the deviation after normalization has
been reduced to +/-8% which is well below the target of +/-15% as
presented previously.
[0054] FIG. 9 is a graph showing the comparison between original
matrix "M" and corrected matrix "M" after normalization with
reference matrix "Mref". The line of equivalence shows the data for
both matrices are essentially the same and the normalization
process is effective.
[0055] For the purposes of this specification and appended claims,
unless otherwise indicated, all numbers expressing quantities of
ingredients, percentages or proportions of materials, reaction
conditions, and other numerical values used in the specification
and claims, are to be understood as being modified in all instances
by the term "about." Accordingly, unless indicated to the contrary,
the numerical parameters set forth in the specification and
attached claims are approximations that may vary depending upon the
desired properties sought to be obtained by the present invention.
At the very least, and not as an attempt to limit the application
of the doctrine of equivalents to the scope of the claims, each
numerical parameter should at least be construed in light of the
number of reported significant digits and by applying ordinary
rounding techniques.
[0056] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in their respective testing measurements.
Moreover, all ranges disclosed herein are to be understood to
encompass any and all subranges therein. For example, a range of "1
to 10" includes any and all subranges between (and including) the
minimum value of 1 and the maximum value of 10, that is, any and
all subranges having a minimum value of equal to or greater than 1
and a maximum value of equal to or less than 10, e.g., 5.5 to
10.
[0057] It is noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the," include
plural referents unless expressly and unequivocally limited to one
referent.
[0058] It will be apparent to those skilled in the art that various
modifications, variations and optimizations can be made to various
embodiments described herein without departing from the spirit or
scope of the present teachings. Thus, it is intended that the
various embodiments described herein cover other modifications,
variations and optimizations within the scope of the appended
claims and their equivalents.
* * * * *