Note: Descriptions are shown in the official language in which they were submitted.
~ WO9~/01413 219~232 r~
A ~I~THOI) l'OR MONfTORlNG PERFORMANCE OE~ AN INCUBATOR MODULE SAID
INC1~BATOR MOi~Ul,E BEING COMPRISED ~' AN AUTO~ATED SYSTEi~l FOR
ASSA~NG IvIUI,TIPLE SAMPLFS AND A lKIT SUITABLE FOR IJSE IN SAID ~qETHOD,
s
Automated systems for csrrying out multiple assays are well known in the art Such
automated systems can incorporate a number of functions for processing assays; incubation,
reagent addition, washing, absorbance Ul~ ~ulrll ~lll, shaking and sample transport The latter
implies that the automated system will basically execute complete assay procedures, without
operator intervention, according to assay protocols present in the systems softtvare, These assay
protocols are, of course, based on the instructions in the test kit package inserts In particular in
the field of biotechnology and e g in the area of immunotechnology large numbers of samples
are to be processed In general in the field of biotechnoklgy microplates are the preferred
containers for carrying out reactions
Good Laboratory Practice (GLP), includes the periodic checkir.g of laboratory
h~ u~ ..LdLiul~ for proper filnr~itl :ng For the majorih! of the functions of the automated
systems for carrymg out multipie assays, suitable means for checking their p~lfiwllldll~,c e~ist An
exception is formed by the incubator modules, said incubator modules bein, used to heat the
samples e g present in microplates to a defined temperahlre and to mainlabl that L~ ,.dLUIC
over a defined period of time
To date the means availabie entail indirect measLIl emellt of the temperature of the samples
at the site of the incubator module Such indirect measurement for e~iample comprises
measurement of the temperature of the l-eating elements of the incubatol module whicll
obviously is a velv crude measul ement of the actual sample temperature and does not take into
accolmt the fact that tl-e module, the sample container and the samples themselves have to wann
up to the temperature sef for the incubator module 'l~either does it enable an accurate analysis of
temperature dispersion over a microplate Arlother n ethod comprises measurement of the
nllL)claLulc of the environment in wi~ich the microplate is present, but also has the same
sholtcomings ~vith regard to warming up and temperature dispersion determillation Anotilel
alternative is to directly measure the temperatul-e of a sample being incubated using a
thermosensor immersed in tl-e sample This is however undesirable due to the fact that for large
numbers of samples SUCh as are present on a microplate an equivalent large number of
IhCIIIU~SCIISUI ~ is required, which would require e.~pensive adaptation of the systems and involve
ulldc~ dl,lc~ increased costs of the system 'f'he impracticality of use of lh~ IUS~ OI ~ on small
SUSSTITUTE SllEET (RllLE 26)
WOg6/01413 ~21 94~32 E~
samples such are often used in assays of biotechnological nature stems also from the interference
with the incubation temperature and the assay itself due to the presence of the sensor itself The
sample si~es are so small that heat transport through the metal sen.sor itself wili influellce the
temperature of the sample. Thus even determination of the lcul~lcld~ulc in one sample present in
a microplate could not be considered accurate.
A p~,lrul~l~a~e check for an incubator comprised in an automated system for assayling
multiple samples ~ 1 r.~ ~y a~ a defined tCIUiJ~.dlU~C in particular suitable for asaying smali
samples requires a medium and means, compatible with the microplate t'vrmat, t'hat will allo~v
qu1ntif~fi~m of the temperature and in particular the ~el~l~ c.d~ul~ distribution during incubation
atthesiteofincubation Inordertobeabletodetectinadequatepclfol,.lall.,coftileillcubatorin
the system the method should have an accuracy better than I ~C and a precision better tha
0.1 ~C.
A Iiquid with ~1.~,. II~UUIll UIIIIC properties would be most suitable as a solution as this woLIkl
allow the photometer of an automated system for carrving out assays such as the ~licroplate
Processor 3û00 (~IE'Pl to be used as a means of ~ ,a;l~ linking absorbance to
temperature. As a literatule search, aimed at finding ru~u~uld~ious for such a liquid, did not
render am. usable option, an alternative was investigated; A buffer solution with a large acidity
(pH) dependence on tèmperature (T) and an acid-base indicator with a large absolbance (A~
dependence on acidity (pH) The result is what was aimed for: a solution of which the
absorbance spectrum is Lcl~ .ldtulc dependent.
Tne subject invention is directed at a method for obtainh~g the desired accuracy and
precision with regard to the actual temperature of a sample in an mcubator in an automated
assay system and even with regard to the temperature dispersion over a number of samples being
simultanevusly incubated In particular the invention is directed at obtaining this hltvrmation in
~5 systems directed at automated processing of microplates. The subject inventioll has the
additional advantages that not on]y the previous hl~ regarding thc techn;cal aspects are
overcome but tllat the solution is simple and relatively low in cost, simple to carly out and
requires little adjustment to e~isting automated systems
Il1e subject invention is directed at a method for mollitoring perfonnance of an incubator
module said incubator module being suitable for incubating multiple samples simultaneously at a
defined ~ J..I~UIC (e.~. for incubating microtitre plates). said incubator module being
SUbSTllUi~ ShEi-r il~.ULE 2b~
WO~C101413 21 ~:2~ P
comprised in an automated system for assaying multiple samples optionally in multiple
~, ,ln "~ assays e g in multiple microtitre plates, said method comprising
1) measuring the absorbance of a Ltlll~ucildLUl~: dependent solution in a photometer (phot 2) at
at least two different ~ dc~ ;Lo il~l ahd ~2, wherein the absorbance of the 1~ ,dLule
dependent solution is determined at a location different to the location of the incubation
and at a moment in time subsequent to the incubation, said L~lll,u~" dLul e dependent solution
comprising a buft'er with a temperature dependent pH and an acid-base indicator w~ith an
absorbance spectrum linked to pl~ and said W I~C~ LLlli~ being selected such that oA/oT is
positive for one wavelength and negative for the other, said measuring providingabsorbance values Ate~phal2~1 and ALes~pho~2A2l wherein AlesLpho~ 2,i.1 indicates the absorbance
(Ai determined for the temperature dependent solution (test), using the photometer (phot
2) at wavelength ~.1 and A~e~phll~ 2,i,2 indicates the absorbance (t'~) determined for the
~a~ UI 1: dependent sohltion (test), using the phu~oulctc~ (pilot 2) at wavelength ~2,
2) saving data of step 1~) in a data file
3j measuring the absorbance of a ~t~ ...... alulc ~ calibration solution for the two
wavelellgtlls ?.1 and ~2 of step 1) in the photometer (phot 2), said t~ul~"dLul~calibration solution exhibiting the same absorbance spectrum as the
~C~ ld~ul~ dependent solution at the defined It~ "d~UIC, said measuring providing
absorbance values A~l,pllal 2.i 1 and A~l.ph~l 7.~.2. Whereill ~4~1,pha~ 2.i 1 indicates the absorbance
(A) determined for the LellllJ~ dLul~ incl j~"d~ ~ calibration solution (cal), usin; the
photon eter (phot 2) at wavelengtTl i~l and ~ P~ . indicates the absorbance (A)
determined tor the temperature ;"dctlclld~,ll calibration solution (cal), using the
photometer (phot 2) at wavelellgth ~2 and
4) saving the data of step 3) in a data file
2~ 5) ca]culating the actual temperature T of the tempc ratul e depelldent solution using the saved
data and the formula
In (l~AlosL~pllot2~Al/Atc5l~phal 2,A?) = O + 13 T,
whereino=o-ln(A,,I,llhall)l/Ac,lphall~2)+1n (Aa~l,ph~2.~ l.pho~2.i.2) withthevaluesof
o - In (Ac~l,phall"~ I,phall.72! beinL~ either p~eLLcl" ;, c i or else being obtainable by
subjecting the ~eul~)cldLul~ independent calihratioll solution to absorbance medsLllclllellL on
a further photollleter (photl) for the t- o wavelellgths ~.1 and ~2 of step 1) thereby
obtainhlg t~,lpha, Iil and Aall.phal 1.~.2. wherein A,lpl"~ indicates the absorbance (~A)
SlJ6STi LJTE SHEET ~fi~lLE 26!
W096~1~13 21 q423~ r ~ s~ c
determined for the ~tjlll~ Ultj; ~ calibration solution (cal) using the turther
photometer (phot 1) at wavelength ~1 and A~lphol ~ 2 indicates the absorbance ~A~
determined for the lcu~ Idlult in~p~n1~nt calibration solution (cal) using the further
photometer (phot 1~ at wavelength ~2 and saving the data of step 5 in a data fiie and
S suhjecting the ~t U~ Ult dependent solution to absorbance and 1~ lulrJ
nl~tt~ulrlllC 1l on the further photometer (phot 1) for the two different ~ . glll~ ~1 and
A2 of step 1 ) thereby obtaining absorbance values A, ,rh~ and 1~ t ph"t 1 A2. at l;nown
~rd~ul j~ from which a' and i3 can be calculated using the forrnula In (Al~/A~2) = a
B T and saving the data in a data file
10 6) comparing the amended Lc u~ d~ e or amended L~ Jwd~ul ~: distribution of a temperahlre
dependent soiution incubated in the module Yv'i~h ~he defined temperature the module is set
to ascertain
Using the raîio of absorbance l l~ ~L~ul~m~ l~ at two dit'ferent wavelengths instead of using
absorbance measurements at a single wavelength offers some interesting advantages The
relation between the s;gnal and the temperature can be expressed as Ihe i'orlïlula 2
In(Aj llAA2) = (~" - a~2) s- (Bj, - Bi 2) T = a' + 13 i' (2)
~ hen one wavelellgtil lies in the range with a positiie oA~oT value and the other in the
range with a ne ~ative oA~oT value using the ratio of t-vo absorbance measurements has the
following advantages
- Increased sensitivit~ of the method
- Increased accuracy of the method since errors caused by ditterent light-path lengths of
samples assa~ed in photometer (phot ]) and photometer ~phot 2) and thelefole errc~rs
caused by i olumetr rc diftel ences in case of a microplate~ are eliminated
- Increased accurd-y of the system since errors caused by differences h~ J~ ,OhOI~
.o"ceia,~lh~ are eliminated
In Clinical Chemistr- 391~' 51-256 (1993) Schilling et al descritbe the use of
thermochromic indicat(Ir solutions such as TRlSlCresol Red in determinin,g the temperature of a
multicuvette photometer in comhination with a thermically hldependerit solution of Cresol Red in
HEPES and phosphate buft'er in a manner to elimmate errors of optical med~ult In~UtS caused by
Yariations of pathlength and blank L ~ e of the wells l~oweYer thev wet e not faced with
SU35T~ T UTE S~iEET IRULE 26)
~2~ ~4232
WO 96/01413 P~ ~
the subject problem of not being able to determine the absorbance at the location of the heated
sample. They carried out their absorbance ~ ul~lucillLs at the location of the It~ .dLult;
~ a~u~ llL i.e. the sample is measured by the photometer at the site of the temperature
d~,tclll hla~iull. They used an electronic probe in one well in order to calibrate the photometer.
Furthermore a third wavelength was required to eiiminate errors produced by different blank
c in addition to the quotie.nt of two wavelengths to eliminate the errors due todifferent light path leng~hs or ~ c~tJl~o~e dLiUUs The temperature h~ L .,1 buffer
is used in the cited literature to simply study the influence of t~huLulll~;l ic noise on the accuracy
and precision of the method and not for calibration purposes as in the subject method. No
problem ~ith regard to cooling down efl'ect is present hl the cited method as it is not directed at
a process similar to the subject method. Simply carrying out the described me~hod of Schilling et
al in the subject situation will not solve the prob1em the subject method solves.
Once the system has been properly calibrated with the method according to the invention,
it aliows d~ ,. ' ' of the Lclll~..dLul~ of the liquid in the wells of a microplate e.g. while
the microplate is in an incubator module of an automated assay system comprising the incubator
module A suitable example of a system in which the method according to the invention can be
carried out is the Microplate Processor 3000.
Based on a literature search, Tris~hydroxymethyl)-d,l,il,u,l.~iL~ ,.c (TRIS) in ~,o~lllJilldliull
with hydrochioric acid (I ICI) was selected as an extremely suitable example of a buRer system to
be used in a method according to te invention. This is mainly hecause of its large op~/oT value
of approx -0 03 pH Wlits/''C. Preferably the but1'er system of the temperature dependent solution
has a large opi ~Ic,T, preferably at least an absolute value of 0~02 pH units /~C'. 1'he larger the
absolute value the better any smali change in temperature w ill register as a noticeable change in
pH leading to a change in the absorbance which is measured, thereby increasing the sensitivity of
the method. In line with this a temperature dependent solution exhibithlg a large ci.~/opH vahle is
preferled i arge can suitably be quantified as a value sufficient to result in at least a precision of
0~1 ~C and an accuracy c~f at least o,5 CC, preferably of at least 0,3 ~C. A number of buffers
other than Ti~lS are also suitable tor use in a method according to the inventic~n Suitable
examples comprise an aqueous solution selected frûm c~trate, tartrate~ phtalate, phosphate~
tris(itlydroxynnethylja,ui,lom~Lhdlle (also i;no--n commonly as TRiS), Borax and sodium
bicarbonate.
SU6STl'rUTE SHEET ~hULE 26)
WO 9~ 413 ~ 3 2 r~
A number of acid-base indicators can be used in Lt~ "d~UlC dependent and temperature
rll 1l solutions for the subject method Ihe selection of an appropriate indicator will be
obvious to a person skilled in the art after considering the invention in the light of this
description The solubility of the acid-base indicator in the temperature dependent and
Lt~l~Jc,dlu,e; 1 ~ 1~,1 calibration solution must be sufficient for measurable and rel;able
ahsorbance values In general currenl apparatus can teliably measure ~ba~ b~ln~.G~ higher than 0,5
AU, so an indicator ., ~ .udLiull leading to such an absorbance at the defined temperature and
at ~a~ and ~~ ;S acceptable A preference for a ~ d~iUII leading to an
absorbance between 0,5 and 1,5 AU is preferred Out of the ac;d-base indicators applicable for
the pH buft'ering range of TRIS, Cresol Red was selected, mainly because of its good solubility
resuitillg hl large oA/opH values Earlier invc~iy,afiuns [1], using ~ ol~,l,Llalein as a pll
indicator e.g. gave poor results because of the poor solubility of pl~- .,n,ll~ ;n TRlS buffer.
Calibration solutions that ha~e Ltln~J., dlu,~ infleppndfnt spectra being identical to the
spectra of the temperature dependent solution at certain Itll~ dLu ta offer some htteresting
possibilities Firstly, such calibration solutions offer the possibility to cAIlrlilll ILallty deterrnine
the precision of the method Secondly such calibration solutions offer the possibility to use
dift'erent photometers for calibrath1g the method (d~,L~ dLiull of c~ and ~ using measured
temperatures and absorbance values) and for actual ~elll~ltldLul~ aultlll ILS Icalculate 1 ~ith
IAnown a and 13 and measured absorbance values)
Usin~S a L~ clLult i (1 ~ calibration solution to compensate for the differencesbehveell phuLu~n~ttl a works as follows [41
The temperature to be measured must have a certain expected value A calihratic~ll solution
must then he available that has a spectrum approximately identical to that of the ternperature
dependent solution at that expected ttlni)cldLult Now the temperature dependent solution is
calibrated (a and B are deten1Iined) using photometer (phot 1) Also the absclrbimce vahles of
the ~ , dLult jn 'epl n~len~ calibration solution at the two wavelengths of interest are measured
on pho~ometer 1 (~I,phlll],; l and ~,I phol~ ;2i
The second photometer is used to measure the absorbance values at the two v,aveltngths
of interest of both the ttlU~J~.dLu~ dependent (Alc~ph~l2 jl and AlCu~p~ l2i~2) and temperature
i,~ calibration solution (Ac,~,pho,2ll and A~lphol~A2) Now the Ul~.a~lJIel~ ia of the
temperature dependent solution from photometer can be expressed hI absorbartce units of
photometer 1, i e the corrected absorbance values, for both wavelelluths
SUoSTlTUTE SHEEI (RlJLE 26)
~ WO~16101413 2 li 94~32 1~
Aco~ = (Ac l,phOn/Ac~l.phoi2) . A~cc~ph~2 (3)
By using the corrected absorbance values the actual It,.~ .dlu~e can now be calculated.
S Combining equations (2') and (3) gives:
In(Alt~t~phol2J ll~iecLF~ 2~2) ~ + 13 .'1 (~)
witl~
0 = A - In(Acoi~phou,~ ,pholl i 2) + In(Ac~srhol2 l llAc~l~phol2 b2) (5
So, in fact, al is re-calibrated into ~"
This method ~ P~ for:
- Small differences in blank media, e.g. water versus air.
- Differences in light-path lengths.
- Small spectral differences in optical ;~ltt~ fi~ cP, filters (Dependent on the value of oL/o~
at the wavelengths used).
As described in literature [21. calibration solutions as described above exist. in the case of a
TRlS/Cresol Red temperature dependent solution the TRTS in the TRTSfCresol Red solution is
replaced by a mixture of EEPES and pilosphate. 13y setting the pH of the calibration solution, its
spectrum can be made approximately identical to that of the TRTS/Cresol Red soiution at any
ta,~,t;, dlui ~ in the range of interest, tnereby enabling the product~orl of t~mi)~" dlUI t~ irrif pf r~ nt
calibration solutions.
As a container comprising l~m~ lul~ dependent solution e.g. a microplate filled with the
temperature dependent solution such as TRlSJCresol Red solution cools do~vll during the
transport frc.m the incubator module to the photometer ~phol 2), (also referred to as the reader
module in the following text) the obtahled a.bsorbance values do not accurately represent the
Lt~ tldiult.> as they were when the microplale was actually still in the incubator module.
Therefore in a method accordhlg to he invention calculating means are used to correct the
absorbance deteremined in the photometer (phc t 2) with regard to the cooling down effect tha!
occurs between the locations of incul ation and absorbance ~IlCdSUl ~ ellt. A ~vay to minimise the
SUibSTlTUTE SHEEI ~P.ULE 26)
wo 96/nl413 2 1 'J ~
inaccuracy is to have the photomeler as close to the incubator as possible and preferably in an
atmosphere as close to the temperature ofthe incubator as possible. A way to o~ercome
inaccuracy due to the transport is to registrate the coo1ing do~n curve for the sample in the
container, preferably for each well of a microplate if a microplate is used and to use these curves
to e.stimate the Le.l~J~. dLUlo':~ as they were in the incubator module.
The subject invention is therefore aiso directed at a method as disclosed above. ~-~herein a
first micropiate filled with temperature i...i. ~ 1 calibration solution is transported to the
photometer (phot 2) and the absorbance is read at the two wavelengths ~1 and ~2, measurement
data is saved in a data file and the microplate is taken to the output module to be removed, a
second microplate comprising temperature dependent solution is taken to the incubator module
and incubated at the defined temperature according to the setting of the incubator modulc and
after completion of incubation is transported to the photometer (phot 2), where the absorbance
is read a number of times at the hYO wavelengtils Al and ~2 at time inters~als pleterably
controlled bY a sofh~are timer and all data is sas~ed in a data filc. In such an cu.L,od;...cul the time
inter~al between incubation termination and the first reading in the photometer (phot 2) is
preferably as short as possible. It is limited by the time required for transport of the microplate
from incubator module to the photometer (phot 2)~ said time interYal preferably being less than
45 seconds, suitably being 25-~S seconds.
A suitable elllbocl;~ ,.lL of a method where the absorbance is read a number of times at the two
wavelengtlls after the container with the L~ .diult: deependent solution has been removed
from the incubator is a method ~Yherehl the time inters~al between absorbarlce measurements in
the photc~meter (phot 2) is determitled by the length of thne recluired for the ~-,~as~" ~u~ of the
absorbance at the two wa~elengtlls ~1 and ~2 and the length oftime required to sas~e thc data hl
a file, said time interval in general being longer than 25 seconds, pret'erably being less than 45
seconds and suitabiy being 30 seconds.
In a method according to the invention, wherein the L~ cllult: determined on tlle basis
of absorbance data S~rom the photometer (phot 71 is corrected ror the COOIhlg down til~lt OCCUIS
betwee,n transport from incubator module to said photometer calculating means on the basis of a
,.,AII,r~,,An..,~l function fitting the pattern of cooiing down such that the regression coefSicient R2
is equal to or larger than 0,9S can suitably be used. The calculating means employefi can for
example enable a function for absorbance against time to be fitted in the least squares sense on
the absorbance Ill~.d~UlS:~lla,lll data obtained in the photollleter (phot 2) enabling calculation of
SUo~TlTUTE SHEET ~ '.ULE 26)
~ WO96/û1413 ~!1 9~3~ r~
actual absorbance in the incubator module at the moment of tal;ing the temperature dependenl
solution from the incubator moclule i.e. at time zero thereby enabling subsequent calculation of
the actual It~ J.~.dLul~ in the incubator module at time zero.
More specifically for the cooling down of microplates the follo~ving was ~ . i ~' The
S tc".~ dlu,e T of an object with an initial ICIII~ dlUI~ Tinl~ that is placed in an environment with
constant t~ U.,. alul e Tc,,~. as a function of time t is theoretically given by:
T(t) = T~n~ + (T"", - Tel~) . e ~T (1 jS a time constant) (6)
I0 However7 for a microplate that cools down, things are much more complicated since the
environment is not constant and differs per well ln fact it is almost impossible to have a
theoretical model for this. In practice~ absorbance values in.stead of Lt:u~ ldlults are measured
as a function of time. It was decided to use a second or thJrd order polynomial as a model for the
absorbance - time relation instead of applying a model for the ternperature-time relation. This
was ;~ fJ using a function like:
A(t) = a + b . t + c . t2 (+ d . t3) (a, b. c and d are constants) (7)
By ftting this model in the least squares sense on the measurement data, a is an estimate for the
absorbance value at time zero.
When multiple samples are ~imlll~neoll51y incubated as is the case for microplates the
It.~ ,. dLU~ t: of sample in each well is inQuenced by neigllbollring wells The influence will vary
with location of the well. In order to increase the accurscy of the method according to the
invention data filterin g can be apphed in the calculations.
The incubator modules of the automated assaying systems such as the ~licroplate
Processor 3Q00 have low frequency characteristics, i e the temperature distribution over the
microplate c.an show itiull,.,.dlule gradients. cold areas, warm areas or an edge effect.
Temperature values jumping up and do~hn from well to well (high frequency behaviour) is not
possible. This implicates that any high frequency compcments in the temperature distribution
obtained by the method hl this report, may be filtered out by means of a so-called ''two
dimrnsional low pass" filter.
SUSSTlTUTE SHEt- l ~;.LILE 26)
~1 6A''
WO96/01413 L I :~'~ JL .
Many commonly used data fil~ering techniques are based on mullip~ ti-~n with an appropriate
function in the frequency domain (w;th the Fourier transform of the signal) or taking the
convolution of the signal with an appropriate function. These techniques require endless .signals
or at least signals that are defined over a relative large area. Sillce for the ~w"~ ule only a
lirnited number (8 x 12) vahles are available in the case of a microtitre plate, this method of
filtering cannot be applied especially because reliable filtered ~,alues for the temperatures al the
edges of the microplate, that are most interestingly, cannot be calculated.
An alternative was 1..~ ,ligaled namely to define the temperature ot'each well as a function
of the tr~ )el dLUI 5,:~ of the well itself and the k~ lu~ ~ of the 8 wells closesl to that well~ e.g.:
~filtD6 = fi . TCs + f7 . Tc6 + f3 . Tc7 + f4 . TDS + fs . ID6 +
-, f~, . Tr~7 + f7 TE5 + fs. TE6 + fs. TE7 (8)
18 Where Th~, is the calculated l~ul~cl~llul~ after filtering and f~ through f~ are constants, 'I'he
con.stants are calculated by fitting a linear model in the sense of the least squares to the
cu~ ul e data of a square block of 9 wells. (two dimensional linear regression~
l~lodel: T~", = a.rownumber + b.~sl"",- ,.,ll.~. + c (9)
~a~ b and c are constants)
Minimizirl2 in the sense of the least squares resu]ts in minimizing fimction F
F = ~ (T,~ wellj) - T~well;))- (i= l .. 9) (I0)
2~
Which means that oF/oa = oF/ob = oFloc = 0. Now a, b and c can be expressed in terms ûf
T(well;) and the t'actors f, through f., can bDe calculated. For a well not at the edge of the
microplate, as in (8), all factors f are equal to 119. For a well at the edge of the microplate but
not at a c.orner, tbe following is valid e.g
~U
f lt,46 = fi .T.~s + f2.T~6 + f,.T,~ + f4.Tg, + fs.TB6 t
f6lr~7~-f~Tc1+fs lc6+t~7l C7 (Il)
SUbiSTlTUTE SHEtT (~ULE 26
WO g6101413 2 ~i 9 ~ 2 P~~
11
With fi = f2 = fi = 5118 fi = f3 = fG = 2/18 and f7 = fs = f~ = -1/18.
It can be noted that the linear regression used to calculate rfiltr~G as in equation (11) is also used
for calculating TfiltR6
For a well at the corner of a microplate the following is valid: e.g.
Tfilt ~ = f~.T~ ~ + f2.TA2 + f3.Tg, + f~.T ~l + f5.TB2 +
+ fG.Tci + f7.Ts3 + fs.Tc2 + f~.Tci (12
With f~ = 8/18 f2 = f3 = 5118 fi = f5 = f6 = 2/18 f7 = f8 = -1/18 and f9 = -4118It can be noted that the linear regression used to calculate 7filt ~ as in equation (12) is also used
for calculating Tfilt~2 Tfil~Bi and 'filtB2.
This spatial low pass filtering technique is illustrated in rrigure I in which the temperature
distribution is represented by a well defined function to which Gaussian noise is added. it will be
appreciated by a person skilled in the art that the calculation of the temperature in the incubator
module can be ensured by fitting a 2 J ~ ~iu~ linear model in the sense of the ieast squares to
the temperature data of a matrbt of samples with the above illustrated square of nine wells for a
microtitre piate merely being an example.
Tlle subject invention is aLso directed at a test kit comprising components necessary for
carrying out the invenlion as described above and in the Example Such a test kit comprising at
ieast a con~ainer comprising a tempera~ure dependent solufion exhibiting a~ least an absorbance
higher than 0 5 AU at wavelengths ;~1 and ~2 at the defined temperature~ said t~ J ldlu
dependent solution comprising a buffer syslern with a l~lnpeliltul~: dependent pE~ and an acid-
base indicatol with an ahsorbance spectrum linked to pH anl a container comprising at least one
~t~ln~ dlu~ nd~lll calibratic)n solution exhibiting an absorbance spec~rum identical to that
of tne temperature dependent solution at the defined temperature~ said t~ dLUIc: inr~ opt n il nt
calibration solution preterably comprising a I~U~niJU~iliUU as close to tnat of the temperature
dependent solution as possible. Optimally the solutions in such a kit have a pl~
SUBSTITUTE SHEET (RULE 26)
2~
W096rO1413 r~"~
12
a) in the working range of the buffer systern of the temperature dependent soiution a~ the
defined temperature at which measuremenl is to take place, said defined Le~ ,.alu.~; bcing
wi~hin a desired te~ alul ~ range, preferably being within the range 20-60 ~C,
b') in the indication range of the acid-base indicator st the defined lt,u.~ 7L~fl~ at which
u.~7,.ll 7~ is to take place,
c) sucil that the a7usl~l7lJa.~7~ at the two w.,~h,..t,Lll~ ~.l and ~2 are as close to eachother as
possible at a temperature in the middle of the specified range
d) preFerably as low as possible to prevent C02 absorption
In particular when a number of defined t~u~7~~ldLurc:~ are to be chec,ked on the incubator a
kit will be used as described above complisillg multiple ~ Lul~ "ld~7,7~"de"l calibratior
solutions for a number of defmed Lel,l~ dLu~s, wherein the pH of each lempelature blll.-l,. .,~i~,a
calibration solution is selected such that the absorbance spectrum of the Lt~ .. dLUI ~ iUdc~ ,UllellL
solution is equal to the absorbance spectrum of the temperature dependent solution at t7he
defined lelllilcildtul~ It will be ciear fiom the description of the method that the I~lU,~J.. dLUI~;
dependent buffer can comprises an aqueous solution of citra~e, tartrate, phtalate, phosphate,
Tris(llydroxyrlletilyl)~ ,nv.Jl~dlllle~ Borax or sodium bicarbonate~ preferably of
Tris(ilydrox),methyl!~-tlino771~ths7ne 11 will also be apparent from the description of the method
according ~o the im~entiotl ~hat a i;it wherein the acid base indicator is Cresol Red and is present
in soluble form is a suitabie embodiment. ln sucll a kit the temperature independent calibration
solution can comprise HFPFS, phosphate and a Cresol Red solution and is preierably further
identical to the temperature dependent solution uhich comprises Tris(h~ydroxymethyl~-
a.";".,,~ "~ ,P as buffer and a Cresol Red solution as acid-base indicator. In order to pre-ent
microbial degradation the solutions in the kit are preferably provided with anti microbial agents
2~ ~enerally used inthe art such as azide. Preferably . ;.. ,. - l~ld~l.yd7~ and gentamicirl sulphate will be
used with a view to reoulations in particular countries. The Example provides further precise
details of the solutions that can be present in a kit and an ~;ullJ~Id;.~.llt of how they can be used.
Preferably a kit accordin,~ to the in-ention s~ill comprise the calibration data required using
phot7)metel (phot l~ thereby rendering the practical application extremely simple and
';0 userfriendly,
In genelal the method and kit according to the imelltion in the various embodiments
disclosed can be used ~o chec77~ the temperature dispersion of the incubator module at a number
SLi'SSTlME SHE T (RU E 27~
-
~ WO96/û1413 ~194232 r~ 7c~
13
of different defined ~ dtul~s~ said method or kit requiring a number of l~ ,.dLu~
,.k l calibration solutions equivaient to the nwrlber of different temperatures In
particular an automated assay in an automated system can be carried out whilst the method
according to the invention is carried out
EXA'MPLE
The method according to the invention has been carried out using a Microplate Processor
3000 as automated assaying system comprising an incubator
The follow-ing reagents were used
1'ris(hydroxymethyl)~ ,;"~" n~ (TRIS)
The working range of a TRTS buffer system is from pH 7 to pH 9 As reported hl literature
[4,5]~ the t~lu3~ Lul~ - pH relation is almost linear in this range with dpHldT = -0 03 pM
unitsJ~C for a 01 moUL solution A stock of a 01 moUL (= 12 114 g,'L) TRIS buft'er was
prepared by dissolving l'RIS in NEl'~ class I quality watel
Setting the pH of the solution was done by adding small amo~mts of a high cun~,cl~LI dLi~n
HCI solution (4~10 mol/L) Because of the temperature dependency, this has to be done at a
controlled temperatule, or the desired pM has to be calculated for the actual t~"",c~lu
'' Cresol ~ed
One of the hldication ranges of Cresol Red is reported to be from pl{ 7 2 to plH 8 8 with a
yellow to red colour transition [6] This means that in the iower region ol' the visible spectrum
the absorbance increases ~ ith increashlg aciditv (decrease of p~l), wilereas in the higher region
of the v isible spectrum the absorbance decreases with increasing acidity
A high concentration solution in ethanol ~ as thought to be the most convenient dosage
form for Cresol Red E~or this purpose a stocl; solution ~vas prepared 17y dissolving Cresol Red
(Kodal;j in ethanol (Baker, 96~,/o) in a concentration of IG 0 g L
3 T-lEPES/phosphate solution
For C~!J~ , "y determining the precision of the method and tor calibration purposes,
the need was felt to have a solution with a temperature in~erPn~Pnr absorbance spectrum, but
identical to that ofthe TRlSJCresol Red solution at a certain temperature
SU6STlTUrE SHEt1' (~ULE 26)
W096fO1413 2~ 'J42~2 ~ c
14
As described in literature 1~1. such a solution can be prepared by repl.lcing the TRIS in the
TRlSlCresol Red solu[ion by a mixture of IIEPES and phosphate.
A stock of HF,PES/phosphate solution was prepared by dissolving l~:PE~S (Organon) and
sodium phosphate, monobasic (Baker) in NEN class I quality water in ~w~ a~ of 26mmol/L (= 6.196 glL) and 76.2 mmoUL (= 11.289 glL) respectiveJy.
4. Preservatives
As the method is intended to be used in a commercial product, the TRlSlCresol Red and
calibration solutions have to be stable for quite some time. For this purpose, preservatives have
to be added to prevent microbial degradation. Althougll comlllonly used, sodium azide was not
selected because of restrictions in some countnes. Instead, . ;.,..~ ,yde and gentamicin
sulphate, as used in some ~,o",lu, c of the latest Organon Teknika ~iicroelisa assays, was
used.
A stock solution of..;"" ~ yde was prepared by diluting ~ yde in 96~~o
ethallol t S~Jo methanol (Baker) in a Co.. ~ dti~ of 200 mllL.
C.llll~l..ald~,i.yde ~Merck) and gentamicin sulphate (USBC) were added to the TRIS and
HEPl :Slphosphate solutions up to final ~.u~ Ll d~;on~ of 0.2 m~L and 0 1 ,~L respectively.
The TRlS/Cresol Red temperature dependent and temperature ;.~ calibration
solutions were prepared in the following manner:
Fcr the Cresol Red concentration and the pH setting of the TRlS/Cresol Red solution, the
following was taken into consideration
- pH should be in the worl;ing range of TRIS buftèr in the temperature range of interest
- pH should be in the indication range of Cresol Red in the temperature range of hltere~st.
pH should be as low as possible to minh~ e C02 absorption
- pH setling sho-lld be such that in the middle of the temperature range of interest (at
approx 37 ~C') the absorbances at the two wavelengths being uscd, are approximately
equal.
- Cresol Red concentration should be such that the absorbance values at the hvo
wavelengttls behlg used, are in the range 0.5 - 1.5 AU (optimal working range of the
reader module of the MPP) in the temperature range of intere.st.
SU15S~ITUTE SHErT (i.ULE 26~
~ WO961\1413 ~ 2~2 r~"~7.~
The pH of the stock solution of TRIS buffer with preservatives added was set to 7.80 at
37 ~C. To a portion of this stock solution Cresol Red (from the stock solution~ was added up to
a final uulll.tlllldLiull of 30 mglL to be used for Ill~.a~UlClU~ > on the a~ ometer~ To
another portion Cresol Red was added up to a final concentration of 75 mg/L to be used for
S ul~aalltll~ .d~ on the MPP. It was verified that the addition of Cresol Red did have no
measurable effect on the pH.
Two calibration solutions had to be prepared i.e. one to be used for ~ ,,aLult~
eaaultlll~ul~ with the incubator module set for 37 ~C and the other for 50 ~C inruhq~inn~ For
the preparation of the calibration soiutions two portions were taken from the HEPES/phosphate
stock solution (with preservatives added) of which the pH ~vas set to 7. 80 and 7.46 respectively.
These p~i values correspond to the pH values of the TRlS/Cresol Red solution at 37 ~C and 50
~C respectively. The two portions uere each divided into two portions to which Cresol Red
(stock solution~ was added up to final concentrations of 30 mg'L and 75 mg'L respectively. It
was verified that the addition of Cresol Red did have no measurable effect on the pH.
The uledsult~ ,t procedures were as follows:
Calibration proc.edure
Speclra were recorded in the visible region 400 through 700 nm using a Pye IJnicam
Model PU8700 ~ uphu~ l and polystyrene cuvettes (I cm optical pathlength) ~rith an
internal width of approx 1 cm. The double walled cell-holder of this instrument is comlected to
a I~UI~.IdlUI~ controlled waterbath via tubhlgs and â pump The temperature in the cuvette is
measured by means of a thermocouple~ positioned jus~ above the lighlbeall) generated by the
a~c~ hOlOme~er. The thermocouple is connected to a Fluke Model 27 multimetel equipped
with a l lodel 80TK Thermocouple~ l~iodule. The amount of tluid bl the cuvette is such that Ihe
insertion depth of the thermocouple is approx. 3 mm.
Spectra for the calibration solutions were recorded at room temperature and saved in data
files for further processing. Just before every measulement~ the specîrophotometer uas blanked
against a cuvette containing water.
Spectra for the TRTSiC'resol Red sohltions were recorded in the range room lemperature
to 5 . ~C. For practical reasons~ spectra were recorded during uarming up or cooling doun of
the liquid in the cuvettes instead of stabilizing the temperature in the cuvette for every
Illta~UI tll~,ul.
SU~STITUTE SHEET (PiULE 26)
WO96/01.113 2~ f 32 r~
16
Heating of the waterbath was set in such a way that a temperature raise of approx I "C'
per 6 minutes in the cuvette was achieved, 8y doing so, the error of the measured IGIII;~ UIG
because of the fact that it lakes a certain time to record a spectrum (approx, 20 sec), is less than
0.1 ~C. The ll~r~UlGillGIII accuracy ofthe IL~ ocuut~le system is 0.1 ~C. Spectra were
recorded at approx. 2 ~C intervals and saved in data files fur fùrther processing. Fûr each
recorded spectrum, the IGIII;~ UIe of the fluid in the cuvette was noted, Just before every
aaulGlll~ the spectrophotometer was blanked against a cuvette containing water,
Cooling of the waterbatil is achieved by heat conduction to a coiled tube positionecd in the
waterbath througll which ~cold) tap water runs. The flo~- of tap water was set in such a way to
achieve a temperature drop of ~u~JIu~illldlGl~ I ~C per c minutes. Spectra were recorded in a
similar way as during heating up.
2, Measurement in the Microplate Processor 3000.
These n.~ ulGIllf llla were performed in microplates ofthe hype Greiner, 12 well stripplate,
flat bottom with curved edges. Each well was filled with 100 mi of fluid ~ha~ was pipetted ~ ith a
caiibrated shlgle channel pipette. All fluids were allowed to reach room temperature before
pipetling and pipetted plates were checked for the absence of air bubbles. The optical pathlerlgth
t'or this volume and type of plate is approx. 4 mm. In order tû obtain approxirrLately the sarne
absorbance values as for the a~G~ ot)LuLulllcter witll a I cm optical pathlength, the Cresol Red
~,ullcelllldliull in calibration fluids and TRIS/Cresol Red fluid as used fol the hll'P had a 2,5
times higher Cresol Red c.oncentration. As mentioned above the signal is in~Ppf nrirnt of the
ChrOmOPI1Ore ~ n~GIIII~I;UI~
The used MPP ~vas one of tile S protûtypes conFigured with prototype software, i,r,
created witll 'T'urho C and running wnder MS-DOS. For the G~T..Ihllullla. two protocols were
;/lo~mn~llGd for the ~IP'P that essentially only differ for the incubation te.mperaturc, i e onr.
protocol for 37 ~C incubation and one t'or 50 ~C incubation. 37 ~C and 50 'C are the two
incuhation tC~IltJ~rllUlt:a at which the incubator modules of the T~IPP are to be tested for
accuracy and temperature distribution over the microplate.
Each protûcol perfûrmed the follov~in~ processing steps:
- The first microplate filled with calibration fluid(s) is transported to the reader module and
is read at two wavelellgths (endpoint readings),
SLlPSTlTUrE SHLEi IP.ULE ~b~
~ WO~i6101413 2 ~ ~232 ~ T~
- Measurement data is saved in data files and the microplate is taken to the output module
for removal by the operator
- The second microplate is taken to the reader module and is read at two ~
(endpoint readings) These readings are related to the temperature outside the MPP and
S the It ,ltJ~.dlu e inside the instrument
- The microplate was then taken to the incubator module #3 and incubated for 20 minutes at
37 ~C setpoint or for 40 minutes at 50 ~C setpoint
- After completion o'the incubation, the microplate was taken to the reader module where
the plate was read 10 times at two wavelengths at time intervals controlled by a software
timer Time zero ~ as defined as the moment the microplate was picked up by the transport
module from the incubator plate carria~e Exact measurement times for the first
wavelength are 30, 60, 90, 1''0, 150, 180, 210~ 740, 270 and 300 sec, For the second
wavelenglil these times are 35, 65~ 95~ 125, 155, Ig5~ 215, 245, 775 and 305 sec All
measurement data was saved in data files for further processing
- After completion of all measurements, the microplate was taken to the output module for
removal by the operator
The time interval of 30 sec ~vas chosen because it takes approx, this time to transport a
microplate from an hlcubatol module to the reader module The time needed by the reader
module to read at tuo wavelellgths and to save the measurement data in a data fiie takes
~ u~ t~ly 30 sec as well ~/'ia the RS-232 connection and a l i~N, data fiies were
transferred to a PC for data processing and data reduction using Lotus Symphony,
The following results uere obtained
Termpel-ature - pH relation of'i'Ri~ butFèr
The 0 1 mol'l TRIS bu~Fer with pil = 7 S0 at 37 ''C' ~-~as used The pM ~aiues of this
solution wele measured in the tempen~ture rant~e fiom approx 20 to 53 ~C, On the measurement
data a linear regression in the sense of the least squares was performed resultin~ in
pH('I') = 8 83 - 0 0277 T (T in ~C~ (13)
SU6STiTUTE SHEE~ (RULE 26)
WO96/01413 21 q ~ ~31~ L ~ 5~a ~
18
With R2 = 0 999 and SterrY = 0.0087 which is well below the measurement accuracy of the
pH. The value opH~oT = - 0.0277 (standard error is 0.00016) is well in accordanc.e ~ith the
values reported in literature [4 5].
Resul~s are visualized in Figure 2.
2. ABSORBANCE SPECTRA OF TRISICRESOL RED SOLUTION AS A FUNCTION
OF T E.MPERArruRE
Absorbance spectra uf the TRlS/Cresol Red solution prepared as abo-!e ~vere recorded in
the temperature range from 26 - 52 ~C. The results are visualized in Figure i. What can pe seen
is that in the range 40û - 473 nm the absorbance increases with an increasinr temperature ~13 is
positive) and in the range 473 - 600 mn the absorbance decreases with an increasing
tJ~.dlUI~ (B is ne~ative~. An isobestic (temperature; ..t~ ) point e cists for 473 nm and
local ma1~ima are located at 576 mn and 435 nm.
To get an impression of the vaiues for a and 13 fiom equation (1) as a function of the
w avelength~ these vaiues uere Gaiculated using only the absorbance spectra at 26 ~C and 52 ~C.
(For each wavelength two equations are then available from which a and 13 can be calculated)
The results are visualized in Figure 4. The relevant wavelengths~ for ~vhich optic.ll interterence
f lters are available in the reader module of the MPP are dra n as veltical lines in Figure 4 (40s
450! 492 and 540 nm). With the limitation of only bein~ able to use these wavelengths it is clear
that the best temperature sensitivity. ~UI e~t ;)nd;l g to the grealest absolute value for 13 in
equation (2)! is obtained when using 405 and 540 nm.
The In(A~/A40 ) values as a function of the temperature were calculated and a and B from
equation (2) were calculated by means of a linear regression in the sense of the least squates.
The results are visualized i n Figure 5 and the regression data are:
a = 1 4g39 (standard error is 0.01û8)
B = - 0 0395~3 ~standald errol is 0.00026)
R2 = 0 999
Sterr~ c~ - 0.0093.
Absorbance values of the two calibration solutions were read at 540 nm and 405 nm on the
spectrophotometer The i'ound values were then used to caiculate virtual temperatLIres using
SWSTITUTE S~iEET ~RUEE 26)
~ WO9~/01413 ;~l 942~2~ r~
equation ~2) and the values calculated for o~' and b'. Surprisingly the calculated virtual
lcll,L,~ lultD were a few~ degrees hir!ller than expected, i.e. 40,4 ~C where 37.0 ~C was expected
and 56.6 ~C where 50,0 ~C was expected. Based on the estimated opH/oT value for the
calibration solution; (7.80 - 7.46)1(40.4 - 56.o) = -0.021, new calibration solutions were
S prepared with pH settings of 7.87 and 7.60 respectively. Absorbance ~ a~ulclllc~ on the
. .U,.ll. n . for these new calibration solutions yielded the foliowing values:
A540 = 0.695 AU and A40s = 1.138 AU for the 50 ~C calibration solution.
A54L = 0.997 AU and A40s = 0.943 AU for the 37 ~C calibration solution.
Calculating the virtual temperatures of these solutions using equation (2) and the values
calculated for O!' and b' yielded values of 50.2 ~C and 36.3 ~C respectively, The spectra of the
solutions corresponded very well ~ith the spectra of the TRlSlC'resol Red solution at these
t t l ~ t:l ~l L Li l t ~.
Appa}ently the absorbance spectrum is, beside by the pl-1. also slightly influenced by whether the
solution contains TRIS or HEPES/phosphate.
3. MEAS1JREMFNTS JN T~rE MICROPLATE PROCESSOR 3000
3. I . Cooling down behaviour
Four .A~ were performed with the incubator module of the MPP set for 37 ~C
incubations and one experinnent with the incuhator module set for 50 ~C incubations,
Absorbance values at the time the microplate was still in the incubator module of the i~lPP
were estimated by fitling a second order pob/nomial in the sense of' the least srluares on the
measurement data. Regression data were excellent indicatimg a second order polynoll1ial to be a
good model for the cooling down behaviour. Using a third order polynomial did not SilOW any
improvement.
SterrYc,( va1ues in the regression analysis are u ell below the measuremellt accuracy of the
reader module of the MPl'
Regression data for all t,'~ltl;lll~,.ll~ can be l'ound in 1'able 1 and some typical examples of
the absorbance values as a l'unction of time (llI..ISUlt Illtlll~ and model) are shown in Figures 6
and 7.
SlJSSTl~UTE S~.EET (PlILE 26
wos~7n~4l3 ~,; 4 ;~ :3 ~ ' r~
3.~. Accuracy and precision
The accuracy of the method i5 defined as the error in the calculated average tt~ ul7~
versus the actual average t~ ..d~UI~ of the fluid in the wells of the microplate in the incubator
module. Precision is defined as the obtained variation in successive determinations of the
5 Itlll,U.IdlLllC.
The accuracy depends mainly on the following:
- The accuracy at which a' and B' are determined.
- Degradation of the TRlS/Cresol Red solution and the calibration solutions.
- Definition of the time zero moment for registration of the absorbance - time curve (cooling
down).
Wi1ell the temperature T is calculated from equation (4)~ the variance of the calculated
temperature can be calculated according to:
Var(T) = Lvar(ln(~A54oJA~,os)) + Yar(a") + T2.~i~ar(B') + 2.T.Cov(a",B')1/B'2 (14)
Eiecause the accuracy is based on the average of a high number of absorbance vahles,
Var(ln~As4o/Aqo5)) in equation (14) may be neglected. When it is furtherrnore assumed that the
inaccuracy ofthe al:sorbance lin.~.~Ul~ X ofthe calibration fluids on the sp..~ ,h.,~""eter
may be neglected, Var(c~"~7 will be equal to Var(o') and Cov~:a",73'! will be equal to Cov(al,B'3
Equation I14) then reduces to:
~'ar(T) = [Var(c~ T2 Var(B') + 7.T.Cov(a',B')~B" (lS)
2S U.sing equation (1~) for determinin~ the acalracy of the method gives standard errors in the
range 0.3 - 0.4 ~C dependant on the temperature Standard errors are 0.31 ~C' at 70 ~(', 0.37 ~C'
at37~CandO.43~C at50~C.
Degradation o~ the solutions has not been investigated It can only be stated that no
obvious degradation effects could be observed in the MPP experimellts over a period of two
~0 months.
Inaccuracies in the determination of time zero of the cool down curve would lead to a
systematic error that can be corrected by redelining time zero base~i on a number of ~A~ x
SULSTITUTE SHEET ~;WLE 261
~ WO96101413 2 1942 ~2 r~
Thanks to the, temperature h~lc~ t~"l calibration solutions, the precision can vely well
be .,~ "h~ L~tly determined A 1 1 mixture of the two calibration solutions was prepared and
two microplates ~vere filled with 100 tll in each well Both plates w-ere read at 540 nm and 405
nm in the reader module of the h,IPP The virtual It.ll~ lulca of the wells of one plate were
S calculated using the other plate as a calibration plate This experiment was performed twice In
first instance, a" was calculated using the average of the absorbance values of all 96 wells of the
calibration plate Not being satisfied with the results, the effect of calculating a" per row of
twelve wells and per individual well has been studied, as well as the effect of applying the spatial
low pass filter on the calculated virtual tc~ "dlu~es Results can be found in Table 2
From the results in Table 2 it is clear that there is a dramafic difference in the precision
(standard error~ for a" being calculated based on the average absorbance of all 96 vvells and
based on the average absorbance of each row of twelve wells The largest relative effect of
applying spatial low pa~ss filtering is seen for cx" being calculated based on the absorbance values
of each individual well This makes sense since in that case the calculated ~ )Cld~UlC for each
well is based on four individual absorbance values, resulthlg in the relative largest rnfluence of
photometric and electronic noise from the reader module ofthe ~IPP
The best results for the precision ~standard error) of respectively 0 03 ~C' and 0 06 ~C are
obtained for calculation of i" per individual well and applying spatial low pass filterinu
The reason for the differences in precision based on the calculation method of a'' ~vas
found to lie within the reader module of the MPP Studying the absorbance values of the
calibration plates used in the various c;~)cl;lllcllla leams that there is a relatic)n to the well
location The average absorbance value of a row of twelve ~vells dift'er t'rom row to row and the
average absorbance value of a column of 8 wells ditl'er trom column to column although all
differences seemed to lie ~vithin the Ill.,~i ,UI clllcllL accuracy of the reader module What is more
important howeier, is that tbe rorv dependency seems to be ~ avelength dependent whereas the
column dependency seems to be wavelength h~dct ~,ldnl~ hen takin~ the ratio of the
absorbance values at two wavelenj,ths, as is being done to calculate the te Il~CldlUl~, the column
dependency is duLom~lLi.,dlli compensated for but the row dependency could theoretically even
become worse Table 3 provides data indicating this etfect Only when calculating a" per ro~v of
twelve wells or per individual well, the row depcndency is cornpensaled f'or as well
SUSS~ITUIE SHE~:T ii~liLE 26~
Wo961~1413 ~1 ~ 42~32 r ~
3.3. Temperature determinations
Four ~tJ~ h~ were performed ~vith the incubator rmoduie of the MPP set for 37 ~C'
incubations and c~ne experiment with the incubator module set for 50 ~C infl~h~ti~ln~
In all ~ Ch~ hl~ la~ 48 wells of the calibration plate (columns I throu~h 6) were filled
S with the 37 ~C calibration fluid and the other 48 wells (columns 7 through 12) were filled with
the 50 ÇC calibration fluid.
Unfortunately l~ lul c calculations with ~ calculated per individuai well can only be
performed t'or 48 wells because of the experiment set-up. At the th11e the experimen~s were
performed however the calculation method for o was not expected to play an important role
as the ~ ~. h"~ for dctG, ~ the precision were carried out at a later stage.
Results can he found in Tables 4 through 8. In Figures 8 through 13 the temperature
distribution over the platea in the S Ch~JC;~ lla has been visualized These Figures correspond
to o being calculated per ro ;v. From the data in Tables 4 through S the efi:'ect of spatial 1O- pass
filtering is not clear By comparing Figure 8 i experimetlt I witl1out spatial low pass filtering) and
Figure 9 (same experiment with spatial low pass filtering) the effect is evident. Figures S through
13 show the results with spatial low pass filtering applied.
From the results in Tables 6 through 8 i~ is clear that the best results are obtained by
calculating o per indi~idual well and applyin ~ spatial low pass filtering which is hl accordance
with the c.~ in determining the precision of the method. This hllplies the need ora full
microplate for each calibration solution.
4 DISCIJSSION
All temperature delermination experiments showed a somewhat lower L~ "c than
the average in the Al well region which can ~ery well be explained by the mechanical
2s constnuction of the heating elements. Also an edge effect is particularly Yisible in the 50 ÇC
experiment Also this is likely to be caused by the mechanical constn~ction of the heating
elements.
TaL.ing the accuracy of the method into account it can be stated that the calculated
average ltl~ lulca in all five ~h~)tlhll ~ta are very ~ell in accordance with the set incubation
le~ )cl dlUI cs of the incubator module. The variation Or the calculated average temperature in the
four 37 ~C ./~ hll.~lla is in accordance with the accuracy of the method. As far as the
temperature distribution is concelned there is no reason to suspect any insufticient performance
SUiSTlTUTE SHELT (RL)LE 26~
~ WO9G/~1413 2 ~ q4~ r~llr~ --
23
for 3~ ~C inrnh~ti~nc, taking the precision of the method into acGount. For 50 ~C mcubations
the 1)~ rul Ulai-~e could be considered questionable, especially due to the rather low tUI..~ dlUI ~"
found in the Al well region. Thus illustrating the effectiveness of hte use of a method according
to the in~ention.
S ~s the ~e~ u~l~olometer is blanked against a cuvette filled uith water and the reader
module of the MPP is blanked against air, an error is introduced in the method because of the
absorbance of the microplate itself. Correcting all absorbance measurements in the reader
module of the ~lPP with absorbance lln,~ Ul~ l at a third (reference) wavelength would be
the fully correct way of working. However, at the absorbance levels being used the effect is only
marginal and the introduction of e~tra uled~u~ would infiuence the precision of the
method in a negative way. Besides, by using a" instead of a' in the t~mp~.d~ul~ n~lclll~ti. nc, the
error is already partly corrected.
5. CONCI,USIONS
The objectives of an accuracy better than I ~C and a precision better than 0.1 ~C are met.
The accuracy and precision of the method are only just not good enough to test an incubator
module against its technical !"~;G~L;UU~ but are by all means suf'ficienl to detect serious
incubator def'ects.
The method is easy to perform. Especially wherl integrated in a commercial product,
including the availabilih~ of processing and calcu1ation routines in the Microplate Processor
3000, an easy to perform and unique method for checking the performance of incubator modules
will be available to customers
SUbSTilUTE SHt-:EI j.~ULE 2
WO96~01413 ~, 9~232
24
REF'ERENCES
1. "A ~e",~ u~ indicative liquid for use in Micro El,ISA incubator valida~ion, a feasibility
study.", Bally, R W,, Subject memo 8760/SAH10004, (May 7, 199l)
2. "Multiwa~velen~lh Photometry of Th~; u,o.l-,u,l,.c Indicator Solutions for Tennperature
D~lt "~ in Multicuvettes", Schilling ~. et al, Clin Chem 39/2, 251 -256 (1993)
3. "Optical methods for monitoring temperature in s~ u~llulo~ ;c analysers", O'Leary
T.D. et al, Ann Clin Biochem 1983;20:153-157
4 "Development of an Aqueous Temperature-lndicatillg Technique and its Application to
Clinical Laborator,v Instrumentatioll", Bo~ ie L. et al, Clin Chel1l 22i4, 449-~'iS5 (197fi~
"Buffers for pH and Metal lon Control, Perrin D.D. and Dempsey'~, B., Chapman and Ha]l
Ltd., London, GB, 143, (Ig74)
6 "The Merck Inde~;n, Merck & Co. Inc., Rahway, NJ, USA, 11'1' ed.. monograpll 2583,
(1989)
SUoSTlT~u~E ~iEET tlULE 26)
2~, 9~2~3~
WO 96/~1413 ~ l 7c~
T.~3LES AND FIGUTRES
Table 1. Regression data for absorbance values during cooling down.
Table 2 Virtual tc.. ~ ult d~ l r . -~ ;.. of calibration plates
Table 3. Row and column dependency of the reader module measured with calibration
plates.
Table 4. Temperature determination for a full plale with o calculated per plate.
Table 5. Temperature d..l. 1U for a full plate with ~ calculated per row.
Table 6. Temperature determination for half a plate with a calculated per plate.
Table 7. Temperature deLt....; .liu.. for half a piate with c~ calculated per row.
Table 3. Temperature ~ ,Lc~llhl~ . for halfa plate vvith o calculated perwell.
Figure IAIB. Spatial low pass filtering by means oftwo dimensional linear regression.
Figure 2 p~ - Itlll~ ldLUlC~ relation of 0.1 mol/L. TRIS buffer
Figure 3. Absorbance spectra of TRIS/C'resol Red solutioll at various tclu~cld~ulta.
Figure 4. \havelength dependency of o and M.
Figure 5. Ln(As~ol~4os) as a functior. ofthe temperature tor the TRIS/Cresol Red solution.
Figure 6. Absorbance as a hùnction ofthe time t'or 37 ~C incubation.
Figure 7. Absorbance as a function of the time for 50 ~C' hlcubation
Figure ~. Temperature distribution of experiment I Witi10U~ spatial low pass filtering.
Figure 9 Temperatu e distribution of experhnent I with spatial low pass filtering.
Figure 10 Temperature distribution of experiment 2 whh spatial low pass filtering.
Figure 11 Temperature distribution of experiment 3 with spatial low pass filtering.
Figure 12 Te nperature distribution of experil11ent 4 with spafial low pass filtering.
Fi=ure 13 Temperature distribution of experiment S Witil spatial low pass filtering.
~5
Al . . i
A Absorbance
AU Absorbance Unit
Co~() Covariance
oA/oT Sensitivity; change of absorbance with temperature
GLP Good Laboratory Practice
HCI Hydrochloric acid
SUhSTlTUTE SHE~:T ~P;UEE ?6)
W09G/01413 2 1 9 ~2 :~ ~ P~~
26
wavelength
LAN Local Area Network
MPP h~iGroplate Processor 3000
NEN N.,~L", ' Eenheids Norm (= Dutch Standard Norm)
S PC Personal Computer
pH Acidity,-Log(H~0 activity)
R2 Correlation coeffic;ent
RS-232 Serial ~ standard
sec Second
Sterr Standard error
Sterr'~, Standard errol oi'the esthllated Y
I Temperature in ~C
TP~lS Tri s( hydro:cymethyl)- ," "; ., ., " ,. I 1~, ~r
Var{) ~ ariance
SU6STITUTE SHEET (RllEE 26
W0 961014t3 2 194 2 32~ r~
~ . =
27
Table I Regression data for absorbance values during cooling down.
Experiment 1 2 3 4 5
Set ~tlll,U~ UlC 37 37 37 37 So
Average A~=o 0.900 0.871 O.g90 0.869 0.631
Average R2 0 999 0 99g 0 99g 0 999 0 999
Minimal R2 0.984 0.997 0.991 0.995 0.999
540 nm Average SterrY,~,O.OOIS 0.0014 O.OOlg 0.0017 0.0013
Maximal Sterr~t 0.0072 0.0026 0.0042 0 0037 0 0033
AverageA,-0 0.91'' 0.891 0.913 0.924 1.047
Average R2 0.99S 0.998 0.998 0.998 0.999
405 nm Minimal R2 0 979 0.990 0.992 0.994 0.995
Avera~e SterrY,s,0.0016 0.0015 0.0015 0.0013 0.0014
Maxil1lal SterrY~"0.0055 0 0036 0.0033 0.0024 0.0035
SUbSTlTUTE SHEET (R!JLE 26~
WO qCI01413 ~ ii 9 ~
28
Table 2. Virtuai temperature d~ ",;.,aL.blls of caiibraLion plates
Experimant I Experimenl 2
without with spatial withouî with spatial
spatialiow low pass spatial low lo~- pass
pass filtering pass filterfilg
filtering fi1tering
AverageT 44.6 44.6 43.1 43.1
a" maximal DT 0 9 0 7 0 7 0.7
calculateù SDr 0.20 019 0.16 0 15
per plate CVr (%) 0 4 0.4 0 4 O.i
a" maainnal DT 0.6 0.3 0.3 0.3
calculated SDl' 0.09 0.07 0.07 0.05
per row C~V'T (~~~) 0.2 0. 1 0.2 0. 1
a" maxima1 DT 0.6 0.3 0 4 0 l
calculated Sr)l- 0.09 0 Qti 0 Q8 0.03
perwell (3~rl (rJo) 0.2 0.1 C~.' 0.1
SUi~sSTlTUTE S'IEET (RiJL' 2fi)
219~2~2
W 0~6/01413 ~ r~ t :.:
29
Table 3. Row and column dc,uc"(LI,c~ ofthe reader module measured with calibration plates.
n=9 540nm 405 nm 540/405
RowAro~rJpl~te SD A~ow/pl~l~ SD Factor,Ow/ SlC~
Factorpl.t~-
A 1.001 0.006 1.009 0.006 0.993 0.002
B 0.9X9 0.006 0.993 0.006 0.996 0.002
C 0.990 0.006 0.990 0.006 1.000 0.002
D ].000 0.003 1.002 0.0()3 0.99S 0.001
E 1.000 0.007 1.00] 0 007 0 999 0 003
F 1.002 0.003 1.004 0.003 0.9g8 0 001
G 1.005 0.()04 1.002 0.005 1.004 0.001
H 1 013 0.004 1.001 0.005 1.013 0.003
n=6 540nm 405 nm 540/405
Row A~0~/Apn~ SD Aro~JApl~le Sl) FactorO,,I A.,~/ SD
Facturp~
1.02i) 0.005 1.019 0.005 1.00] 0.00]
2 1.006 0 003 1.00~ 0.007 1.002 0.001
3 0.99g 0.007 O.g99 0.007 O.g99 0.001
4 O.g95 0.006 0 g97 0.005 0.999 0.001
09Sg 0.009 0.9g9 0.009 0 99S 0.001
6 0.99CJ O.OOg 1.000 0.009 O.g99 0.001
7 1 000 0.006 1.00~ 0 007 0.9g~ 0.001
X ].003 0.005 ].~)04 0.005 0.9g9 0.002
9 0.99& 0.005 0.99S 0.005 ].00] 0 001
0.992 0.004 0.992 0 004 1.000 0.002
Il 0.9gg 0.010 0.996 0.011 1.002 0.002
12 1.001 O.OOS 0.999 O.OOS l.002 0.001
SUESTITUTE SI~E''I ~PtULE 2~
W096101413 2 i 9 ~2 32 30 ~
c.~ ~, .~ X ~
3c~ 3 ~ o~ ~ G O
~_ V. ~
c-- c . oc ~ ~ O
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3 v o ~ ~t
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c c ~~ ~ ~ o! O
v 0~
O _ C C~ ~ ~ ~ ~
c ~~ 3 v~ ~_ o a~
~: 3 c -- c ~ ~ o o o
c ~ _ v ~1)
-- o _ c , o --
c ~ ~ c
c c~
c -- ~
c -~ c c , -- o
~ ~ v ~0
C C ~, 'D X ~ ~r,
c 3 ~ 1-- 0 0 0
O ~ r~
~ -- o o
c
~ ~
SUISST~TUTE SHEET (RULE 26)
- ~I q~232
~ W096/01413 3 1 r~ rl. .
c c, ~ o o
V OQ
~ ~ ~ c ~-- x
~--3 c 3 -- ~
_ v. o~
v 3 _ ~_ _ O O
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v c~)
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O = v , ~ ~ v~
~ O O
3 c c
v~
a
' 1 ~ ~4 ,~ o
Sl L L ~ - v~ (,
Sll~STITUTE SHEET (RULE 2~)
Table 6 Temperature d~t~, I for half a plate with a" calculated per plate.
Experiment 1 2 3 4 5
n=48 without with spatial without with without with without with spatial without with
spatial low low pass spatial spatial spatial spatial spatial low pass spatial spatial ~ -
~J pass filtering low pass low pass lo-v pass low low pass filtering low pass low pass .
~ filterh~g filtering filtering filtering pass filtering filtering filtering -r
c filtering ~ r~
r averageT 37-5 375 371 37.1 37.0 37.0 374 37.4 50.0 500
37 maximal ~T 0.8 0 7 0.9 0.7 0.8 0.7 0.9 0 8 1.7 1.5
r~ SD~ 0.21 0 18 0.21 0.17 0.22 0.19 025 0.21 0.39 0.35
CYr(%) 0.6 0 5 0.6 0.5 0.6 0.5 0.7 0 6 0.8 0 7
3 2
~ W09C/01413 3 3 r~
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-- _ v ~~
s~3 ~ ~ .~ ~ _ O O
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3 ~ -- o o
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c 3 v 3 3 ~ -- b o
3 ~ 3 _ r~ o o o
v~ O ~~ .
~'I
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3 v O ~ ~ ~
~n ~ ~o o O
3 hO o
3 Q, ~:
~ -- ~c
~
r v~
SWSTITUTE SHEET ~P~ULE 26)
2~ q~2 7l2
~096/01413 3 4 P ~ c~c ~
--3t~ t~ _ O O t~ ~
5 t~ ~ t_ ~ t ~ t ~o
t~ ~) ~I t;~ t~l ~0
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~ 3 V ~ o tlO t~~l V.
t I tJ 2 r' o o o
tl
3 o .~ t t o o~ t'l
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3-- t~ t- )
V.
3 t 3 _ t' O O O
V o ~ t 7
t I
3 c '.~ C ~~ -- t -- u ~
t ~ V ~ t-- -- V
O t~
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t=~ ~tl~ O
3 ~ 2 t
t ~ F
SU3STIT'JTE S11EET IRULE 261
