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`US 20030106682Al
`
`(19) United States
`(12) Patent Application Publication
`Reid et al.
`
`(10) Pub. No.: US 2003/0106682 Al
`Jun. 12, 2003
`( 43) Pub. Date:
`
`(54) SIDE-WALL HEATER FOR
`THERMOCYCLER DEVICE
`
`(76)
`
`Inventors: Taylor Reid, Carlsbad, CA (US); Roger
`Taylor, San Diego, CA (US); Larry
`Brown, Carlsbad, CA (US)
`
`Correspondence Address:
`PALMER & DODGE, LLP
`KATHLEEN M. WILLIAMS I STR
`111 HUNTINGTON AVENUE
`BOSTON, MA 02199 (US)
`
`(21)
`
`Appl. No.:
`
`10/261,751
`
`(22)
`
`Filed:
`
`Oct. 1, 2002
`
`Related U.S. Application Data
`
`(60)
`
`Provisional application No. 60/326,599, filed on Oct.
`2, 2001. Provisional application No. 60/346,114, filed
`on Oct. 19, 2001.
`
`Publication Classification
`
`Int. Cl.7 ........................................................ F24F 3/00
`(51)
`(52) U.S. Cl. .............................................................. 165/206
`
`(57)
`
`ABSTRACT
`
`The invention relates to an apparatus and methods for
`preventing condensation on the interior surfaces of sample
`tubes which are being exposed to temperature cycles, such
`as during a PCR amplification reaction. In particular, the
`invention relates to apparatus comprising a sample block
`comprising a plurality of sample wells for receiving sample
`tubes and heating elements disposed in the sample wells for
`heating at least a portion of the sides of sample tubes (e.g.,
`at least the portion which forms the head space after a tube
`is filled with a PCR reaction mixture). In a preferred aspect,
`the sample block is part of a thermocycling device for
`performing PCR and the side-wall heater is used to enhance
`uniformity and speed of amplification reactions. For
`example, by decreasing or eliminating condensation, signal
`strength jumps in a real-time PCR assay can be minimized
`as can reaction non-homogeneity.
`
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`Agilent Exhibit 1231
`Page 1 of 19
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`

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`Patent Application Publication
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`Jun. 12, 2003 Sheet 1 of 9
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`US 2003/0106682 Al
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`Agilent Exhibit 1231
`Page 2 of 19
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`Patent Application Publication
`
`Jun. 12, 2003 Sheet 2 of 9
`
`US 2003/0106682 Al
`
`Agilent Exhibit 1231
`Page 3 of 19
`
`

`

`Patent Application Publication
`
`Jun. 12, 2003 Sheet 3 of 9
`
`US 2003/0106682 Al
`
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`Agilent Exhibit 1231
`Page 4 of 19
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`Agilent Exhibit 1231
`Page 5 of 19
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`

`

`Patent Application Publication
`
`Jun. 12,2003 Sheet 5 of 9
`
`US 2003/0106682 Al
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`
`Agilent Exhibit 1231
`Page 6 of 19
`
`

`

`Patent Application Publication
`
`Jun. 12, 2003 Sheet 6 of 9
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`US 2003/0106682 Al
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`Agilent Exhibit 1231
`Page 7 of 19
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`Patent Application Publication
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`Agilent Exhibit 1231
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`Page 10 of 19
`
`

`

`US 2003/0106682 Al
`
`Jun. 12,2003
`
`1
`
`SIDE-WALL HEATER FOR THERMOCYCLER
`DEVICE
`
`RELATED APPLICATIONS
`
`[0001] The present application claims the benefit of U.S.
`provisional application with Serial No. 60/326,599, filed
`Oct. 2, 2001 and U.S. provisional application with Serial No.
`60/346,114, filed Oct. 19, 2001, the entirety of each is
`incorporated herein by reference.
`
`FIELD OF THE INVENTION
`
`[0002] The invention relates to a side-wall heater for use
`in a thermocycler device.
`
`BACKGROUND OF THE INVENTION
`
`[0003] Polymerase chain reaction (PCR) exponentially
`amplifies DNA using temperature cycling to generate mil(cid:173)
`lions of copies of a target nucleic acid sequence from limited
`starting amounts of nucleic acids. Generally during PCR,
`target DNA is amplified by denaturing the DNA, annealing
`short primers to resulting single strands at specific sites (e.g.,
`sequences flanking the target site) and extending the primers
`using a thermostable polymerase to generate new copies of
`double-stranded DNA complementary to the target. Typi(cid:173)
`cally, the PCR reaction mixture is repeatedly cycled (e.g.,
`20-50 times) from high temperatures (e.g., >90° C.) to
`denature the DNA to lower temperatures (e.g., between
`about 37° C. to 70° C.) for primer annealing and extension.
`Primer annealing and extension can be performed at the
`same or different temperatures.
`
`[0004]
`In most automated PCR instruments, the reaction
`mixture is placed within a disposable plastic tube which is
`closed with a cap and placed within a metal heat-conducting
`sample block. The sample block is in communication with a
`processor which controls the cyclical heating of the block.
`As the metal block changes temperature, the reaction mix(cid:173)
`ture is exposed to similar changes in temperature. Generally,
`PCR instruments provide a heating element at the bottom of
`the sample block in the form of a Peltier thermoelectric
`device or a thin foil heating element (MINCO brand, Min(cid:173)
`neapolis, Minn.) or alternatively supply a heated or cooled
`fluid through channels machined into the sample block. The
`use of these types of heating devices can result in delays in
`transferring heat from the sample block to the reaction
`mixture which may not be the same for all samples. Thus,
`both the efficiency and uniformity of amplification of
`nucleic acids within the samples can suffer as a conse(cid:173)
`quence.
`
`[0005] Evaporation from the PCR mixture during thermal
`cycling also can decrease the uniformity of amplification.
`Since the reaction mixture generally occupies only a fraction
`of a sample tube, this leaves a volume of air (known as "head
`space") above the reaction mixture into which the reagents
`of the reaction can evaporate and subsequently condense.
`Various strategies have been used to minimize this problem.
`For example, a hydrophobic material such as mineral oil can
`be layered onto the PCR reaction mixture. The hydrophobic
`material floats on the reaction mixture and completely
`covers the surface of the reaction mixture, preventing evapo(cid:173)
`ration from the mixture and condensation onto the side walls
`of the sample tube. A variant method relies on the use of a
`small solid wax ball or grease that melts at denaturing
`
`temperatures and which can be used to cover the surface of
`the reaction mixture (see, e.g., as described in U.S. Pat. No.
`5,411,876). A commercially available wax ball used for this
`purpose is AMPLIWAX available from PERKIN-ELMER,
`Norwalk, Conn., U.S.A. However, adding hydrophobic
`materials or wax balls to the reaction mixture is both time
`consuming and increases the probability of sample contami(cid:173)
`nation.
`
`[0006] Another strategy to prevent or minimize sample
`evaporation and condensation includes the use of an external
`heater which is in proximity with the sample block. For
`example, the Stratagene Hot Top Assembly™ hot top for the
`RoboCycler® thermocycler provides a mechanism for heat(cid:173)
`ing the top of sample tubes placed in a sample block while
`the thermocycler's heating element heats the bottom of the
`sample block. The Hot Top Assembly™ significantly
`reduces condensation but a slight ring of condensation above
`the PCR reaction mixture in sample tubes may still be
`observed.
`
`SUMMARY OF THE INVENTION
`[0007] The invention relates to an apparatus and methods
`for preventing condensation on the interior surfaces of
`sample tubes which are exposed to temperature cycles, such
`as during a PCR amplification reaction. In particular, the
`invention relates to apparatus comprising a sample block
`comprising a plurality of sample wells for receiving sample
`tubes and heating elements for heating at least a portion of
`the sides of sample tubes (e.g., at least the portion which
`forms the head space after a tube is filled with a PCR
`reaction mixture). In a preferred aspect, the sample block is
`part of a thermocycling device for performing PCR and the
`side-wall heater is used to enhance uniformity and speed of
`amplification reactions. For example, by decreasing or
`eliminating condensation, signal strength jumps in a real(cid:173)
`time PCR assay can be minimized as can reaction non(cid:173)
`homogeneity.
`
`[0008]
`In one aspect, the invention provides an apparatus
`for heating an upper portion of the interior surface of a
`sample tube, the upper portion extending from the top of the
`tube to a point above a lower portion of the tube which is
`filled with a fluid. The apparatus comprises a side-wall
`heating block which comprises a heat-conducting material
`(e.g., such as aluminum) and at least one sample hole for
`receiving the sample tube. The sample hole runs from an
`upper surface of the block through a lower surface of said
`block and is alignable with a sample well of a sample block
`for a thermocycler apparatus. The heating block further
`comprises a heating element (e.g., such as a resistive heating
`element) disposed on or in the side-wall heating block. The
`heating element is connectable (directly or indirectly) to a
`power supply for activating the heating element to thereby
`provide heat to the heat-conducting material.
`
`[0009]
`In a preferred aspect, the heating block further
`comprises a temperature sensor for detecting the tempera(cid:173)
`ture of the block. The temperature sensor is connectable to
`a processor for monitoring the temperature of the block, the
`processor being connectable to the power supply and for
`providing instructions to the power supply to activate the
`heating element to maintain the temperature of the block to
`a level which is the same or higher than the temperature of
`a fluid in the lower portion of the tube for a selected period
`of time.
`
`Agilent Exhibit 1231
`Page 11 of 19
`
`

`

`US 2003/0106682 Al
`
`Jun. 12,2003
`
`2
`
`[0010] Preferably, the side-wall heating block comprises a
`plurality of sample holes for receiving a plurality of sample
`tubes. In one aspect, at least one sample hole is conformed
`to fit a sample tube for use in a PCR reaction such that the
`sample tube does not freely rotate in said sample hole or
`there is 1 mm or less of space between the walls of the
`sample hole and the external walls of the sample tube.
`
`[0011]
`In one aspect, the sample tube can receive up to 0.2
`ml of a fluid. In another aspect, the sample tube can receive
`up to 0.6 ml of a fluid. In still another aspect, the sample tube
`can receive up to 1.5 ml of a fluid. In a further aspect, the
`sample tube is connected to a plurality of other sample tubes,
`each other sample tube received by a sample hole in the
`side-wall heating block, i.e., the sample tubes are connected
`as a strip of sample tubes. In this scenario, the "top" of each
`tube extends from the portion of the tube which rests at the
`top of the sample hole.
`
`[0012] The side-wall heating block can comprise any
`number of sample holes. In one aspect, the block comprises
`24 sample holes, 32 sample holes, 48 sample holes, 96
`sample holes, or 384 sample holes. Preferably, the sample
`holes are arrayed as in a standard microtiter plate format. In
`one aspect, the sample holes are arrayed in 8 rows. In
`another aspect, the sample holes are arrayed in three, four,
`six, or twelve columns.
`
`[0013] The invention further provides an assembly com(cid:173)
`prising an apparatus as described above and a sample block
`for a thermocycler comprising at least one sample well,
`wherein any sample holes in the side-wall heating block are
`aligned with openings of any sample wells in the sample
`block. In one aspect, the sample block comprises a heating
`element and the heating element in the side-wall heating
`block is controllable independently of said heating element
`in the sample block. Preferably, the sample block further
`comprises a cooling element. In one aspect, the at least one
`sample well in the sample block is beveled to receive a
`conical portion of a sample tube comprising a conical
`portion and a cylindrical portion. The sample hole of the
`side-wall heating block is conformed to receive the cyclin(cid:173)
`drical portion of the sample tube.
`
`[0014] The invention also provides a thermocycler device
`comprising the assembly described above. In one aspect, the
`thermocycler device further comprises a hot top assembly
`for heating the external surface of the top of the at least one
`sample tube placed in a sample hole in the side-wall heating
`block.
`
`[0015]
`In a further aspect, the thermocycler device is
`connectable to a detector for detecting optical signals from
`an at least one sample tube disposed in the assembly.
`Preferably, the detector enables detection of a signal gener(cid:173)
`ated during the progress of an amplification reaction.
`
`[0016] The invention also provides a method for heating
`the upper portion of a sample tube above a portion of the
`tube which is filled with a fluid (e.g., such as a reaction
`mixture used in an amplification reaction). The method
`comprises placing the sample tube in a sample hole of the
`apparatus as described above, the portion of the tube com(cid:173)
`prising the fluid protruding past the lower surface of the
`side-wall heating block and activating the heating element to
`heat the upper portion of the sample tube. Preferably, the
`side-wall heating block is heated to a temperature which is
`
`the same or above the temperature of the portion of the tube
`comprising the fluid. Heating of the side-wall heating block
`can be used to prevent condensation on interior walls of said
`sample tube.
`
`[0017]
`In one aspect, the portion of the tube comprising
`the fluid is within a sample well of a sample block which
`comprises a heating element which is controlled indepen(cid:173)
`dently of the heating element in the side-wall heating block.
`Preferably, the sample block cycles the portion of the tube
`comprising the fluid through a least one change of tempera(cid:173)
`ture. More preferably, the sample block cycles the portion of
`the tube comprising the fluid through cycles of a PCR
`reaction.
`
`[0018]
`In one aspect, the heating element in the side-wall
`heating block maintains the side-wall heating block at a
`temperature which is the same or above the temperature of
`the sample block for a selected period of time (e.g., through(cid:173)
`out the course of a PCR reaction, such as for at 20-50 cycles
`of a PCR reaction). In a preferred aspect, the heating element
`of the side-wall heating block maintains the side-wall heat(cid:173)
`ing block at a temperature which is above the highest
`temperature through which the sample block is cycling (e.g.,
`at a temperature greater than 90° C., and preferably greater
`than 94° C., where the cycling is part of a PCR reaction). In
`a preferred aspect, the side-wall heating block maintains a
`uniform temperature while the sample block is cycling
`through changes of temperature.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`[0019] The objects and features of the invention can be
`better understood with reference to the following detailed
`description and accompanying drawings. The drawings are
`not to scale.
`
`[0020] FIG. 1A is a schematic diagram of perspective
`view of an assembly comprising a side-wall heating block
`and a sample block for use in a thermocycler according to
`one aspect of the invention. FIG. 1A shows a diagram of an
`assembly configured for one tube; the positions of the tube
`within the heating block and sample block are shown by
`means of dotted lines.
`
`[0021] FIG. lB shows a photograph of an assembly
`configured for multiple tubes according to one aspect of the
`invention.
`
`[0022] FIG. lC shows sample tubes showing no side wall
`condensation after thermocycling with the use of the side(cid:173)
`wall heating block.
`
`[0023] FIG. 2A is a table showing a demonstrating uni(cid:173)
`formity of PCR amplification obtained using the side-wall
`heating apparatus according to one aspect of the invention.
`
`[0024] FIG. 2B is a plot showing the uniformity of PCR
`amplification in different sample tubes placed in the side(cid:173)
`wall heating apparatus according to one aspect of the
`invention.
`
`[0025] FIG. 2C is a contour plot schematically illustrating
`the amplification uniformity shown in FIG. 2B according to
`one aspect of the invention.
`
`[0026] FIG. 3 is a figure illustrating the assembly of the
`side-wall heater according to one aspect of the invention.
`
`Agilent Exhibit 1231
`Page 12 of 19
`
`

`

`US 2003/0106682 Al
`
`Jun. 12,2003
`
`3
`
`[0027] FIG. 4 is a diagram showing the specifications of
`the side-wall heater according to one aspect of the invention.
`
`DETAILED DESCRIPTION
`
`[0028] The invention provides an apparatus for heating at
`least a portion of the upper walls of sample tubes placed
`within the sample wells of a sample block adapted to receive
`the lower portion of the sample tubes. Preferably, the appa(cid:173)
`ratus is conformed in size and shape to fit within a ther(cid:173)
`mocycler apparatus adapted to hold one or more sample
`blocks (e.g., such as Stratagene®'s Mx 4000 or RoboCy(cid:173)
`cler® thermocycler).
`[0029] Definitions
`[0030] The following definitions are provided for specific
`terms which are used in the following written description.
`[0031] As used herein, the term "block" refers to a struc(cid:173)
`ture, usually metal, which can be temperature controlled and
`in which wells or holes have been arranged to accept tubes
`containing reaction mixtures or "samples."
`[0032] As used herein, "head space" refers to empty space
`within a sample tube which has been partially filled with a
`fluid (e.g., such a reaction mixture). In one aspect, head
`space is at least 2/3, Yi, 1/3, %, 9/10, o/s, or 1o/is of the total
`volume of a sample tube. In another aspect, head space is the
`space remaining after a 0.2 ml, a 0.5 ml, a 1 ml, or a 1.5 ml
`tube is filled with 10, 20, 50, 100, or 200 µl of a fluid, such
`as a reaction mixture.
`[0033] As used herein, a "reaction mixture" refers to a
`volume of fluid comprising one or more of a buffer for a
`PCR reaction, one or more nucleotides, a polymerase, and a
`sample containing or suspected of containing a nucleic acid.
`[0034] As used herein, the "upper section of a sample
`tube" or the "upper portion of a sample tube" refers to a
`portion of a sample tube from the top of the tube to
`approximately the midpoint of the sample tube or to a point
`between the midpoint and the top of the tube. In one aspect,
`the upper section is 1 mm, 2 mm, 3 mm, 4 mm, 5 mm in
`length.
`[0035] As used herein, the "top of the tube" refers to the
`section of the tube which extends from the part of the tube
`which rests at the top of the side-wall heating block. In one
`aspect, the "top of the tube" extends from the lid of the tube
`or from the portion of the tube which is capped when a tube
`is capped. However, in another aspect, where the tube is part
`of a strip of tubes (see, e.g., as shown in FIG. lC), the top
`of the tube extends from the strip which connects the tubes
`as the strip will rest at the top of the side-wall heating block
`(see, e.g., as shown in FIG. lB).
`[0036] As used herein, the "lower portion of the tube"
`refers to at least the portion of the tube which comprises a
`fluid such as a reaction mixture.
`[0037] As used herein, a "side-wall heater" refers to a
`heating element for heating at least an upper portion of the
`side-walls of a sample tube or the head space of a sample
`tube, such as a PCR or eppendorf tube.
`[0038] As defined herein, "decreasing or preventing or
`eliminating condensation" refers to a lack of visible con(cid:173)
`densation of the side-walls of a sample tube immediately
`after one or more amplification cycles or a reduced amount
`
`of condensation as compared to a control tube which has
`been subjected to the same cycles of amplification without
`a side-wall heater (50% less condensation, 80%, less con(cid:173)
`densation, 90% less condensation, 95% less condensation,
`98% less condensation, 100% less condensation compared
`to a control tube) such that sample volume remains essen(cid:173)
`tially the same throughout the reaction (at least 95%, 97%,
`98%, and up to 100% of the sample volume does not change
`throughout the amplification reaction). Preferably, no con(cid:173)
`densation is observed after at least at least 10, at least 20, at
`least 25, at least 30, at least 35 cycles, at least 40 cycles, at
`least 45 cycles, or at least 50 cycles of amplification.
`
`[0039] As used herein, the term "cycle" refers a series of
`temperature steps over selected time periods which result in
`the amplification of a target nucleic acid. A cycle minimally
`comprises a denaturing step and a primer annealing and
`extension step. In one aspect, a cycle comprises a denaturing
`step of 90-100° C. (preferably, 94° C.) for 30 seconds-1
`minute (preferably, 30 seconds), an annealing step from 37°
`C.-60° C. (preferably, 55-57° C.) for 1-2 minutes (preferably
`1 minute), followed by an extension step of 70-75° C. for 30
`seconds to 2 minutes (preferably, for 30 seconds).
`
`[0040] As used herein, an "amplification reaction" or a
`"PCR reaction" refers to a plurality of cycles which results
`in a desired amount of amplification or which is selected for
`a desired amount of amplification. In one aspect, a PCR
`reaction comprises at least 10, at least 20, at least 30, at least
`40 or at least 50 cycles.
`
`[0041] As used herein, "real time target template synthe(cid:173)
`sis" or "real time synthesis" refers to a synthetic process
`(e.g., such as an amplification reaction) during which the
`synthesized product (e.g., double stranded DNA) can be
`analyzed as it is being generated without affecting subse(cid:173)
`quent synthesis of the product.
`
`[0042] As used herein, a "sample" or a "test sample" refers
`to any substance comprising a target nucleic acid of interest.
`For example, a sample can comprise a cell, tissue or portion
`thereof, bodily fluid (including, but not limited to: blood;
`plasma; serum; spinal fluid; lymph fluid; synovial fluid;
`urine; tears; stool; external secretions of the skin, respira(cid:173)
`tory, intestinal and genitourinary tracts; saliva, and the like),
`organ or portion thereof; organism (e.g., bacteria) or portion
`thereof. A sample also can also be obtained from a natural
`source (e.g., such as a lake) or industrial source (e.g., such
`as a food product) suspected of comprising a biological
`material.
`
`[0043] As used herein, a height of a side-wall heating
`block which is "substantially the same" as the longitudinal
`length of the upper portion of the sample tube refers to a
`height which varies by less than 5 mm from the longitudinal
`length of the upper portion of the sample tube, and prefer(cid:173)
`ably varies by less than 2 mm from the longitudinal length
`of the upper portion of the sample tube.
`
`[0044] As used herein, a device "connectable" to another
`device refers to a device which is capable of forming an
`indirect or direct connection with the other device such that
`the output of at least one of the devices can be provided to
`the other device. For example, a heating element connect(cid:173)
`able to a power supply can at least receive power from the
`power supply to be activated by the power supply. In one
`aspect, output can be provided to the heating element
`
`Agilent Exhibit 1231
`Page 13 of 19
`
`

`

`US 2003/0106682 Al
`
`Jun. 12,2003
`
`4
`
`through a direct electrical connection. A processor connect(cid:173)
`able to a power supply can, upon connection, transmit
`instructions to the power supply to direct the supply of
`power (e.g., in the form of a current) from the power supply
`to another device to which the power supply is connected
`(e.g., such as the heating element of the side wall heater). A
`processor connectable to a temperature sensor can receive
`temperature information from the temperature sensor upon
`connection and based on this information can transmit
`instructions to the power supply to activate or inactivate the
`heating element.
`
`[0045] As used herein, maintaining a temperature for a
`"selected period of time" refers to a period of time identified
`by a user of the apparatus or by a program which is
`programmed into a processor connected to the apparatus. In
`one aspect, a selected period of time is the length of a PCR
`reaction (e.g., 20-50 cycles).
`
`[0046] As used herein, a sample hole of said side-wall
`"conformed to receive" a particular size or shape of sample
`tube refers to a sample hole whose dimensions are substan(cid:173)
`tially similar to the size and shape of a sample tube such that
`the sample tube snugly fits within the sample tube such that
`the sample tube does not freely rotate (i.e., without being
`manually or otherwise moved) within the sample hole or that
`there is less than 2 mm, and preferably less than 1 mm of
`space between the external wall of the sample tube and the
`walls of the sample hole.
`
`[0047] As used herein, "at least one change of tempera(cid:173)
`ture" refers to a change of temperature which results in an
`at least 5° C., at least 11° C., at least 20° C., at least 40° C.,
`or at least 50° C. change.
`
`[0048] As used herein "maintaining a temperature for a
`selected period" refers to obtaining a temperature which
`remains substantially constant or "uniform" (e.g., does not
`vary by more than 3° C., preferably does not vary by more
`t