CA2194232A1 - A method for monitoring performance of an incubator module, said incubator module being comprised in an automated system for assaying multiple samples and a kit suitable for use in said method - Google Patents

Abstract

A method and a kit for monitoring performance of an incubator, siad incubator module being suitable for incubating multiple samples simultaneously at a defined temperature (e.g. for incubating microtitre plates), said incubator module being comprised in an automated system for assaying multiple samples optionally in multiple simultaneous assays e.g. in multiple microtitre plates.

Description

~ 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 3 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~~

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~"~

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~

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 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~

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 ~

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.

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~ --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 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~~

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~
~ . =

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 ~

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 :.:

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
~-- ~ 3 ~ o~ r~i o 3 v o ~ ~t ~a v 00 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
o c ~r 3 -- ~ ~
.~ c 3 ~3 ~ _ O O
3 ~ O c ~_ 3 ~ ~ ,, o 3 c -- ~ ., .
3 r~
~ o ~ ~ o ~ ~ ~
.--~, 3 ~ ~ -- o o v c~) c _ ~
3~L 3 -- r~ o o o .~ _ -- _ v' . 3 ~ 3 ~ r~ o o o 3 ~ r o o o . _ _ ~

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 ~ W09C/01413 3 3 r~

v u~
3 S:L 3 ~ o ~
v O
u~
~- ~ o ~, ~. ~o ~ " 3 ~ o -- o o 3 o ~

v 3 ~ o o o 3 ~ '~
-- _ v ~~
s~3 ~ ~ .~ ~ _ O O

3v, ,o o 3 ~ -- o o ~ o ~ ~, o -- ~ ~
c 3 v 3 3 ~ -- b o 3 ~ 3 _ r~ o o o v~ O ~~ .
~'I
.. _ _ v u~

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 .-- 3 -- t~
O ~;:

~ ~~ C ~ O t ~ ~' _ ~ 3 t~ b o 3 V. o t~ t l ~ 3 V ~ o tlO t~~l V.
t I tJ 2 r' o o o tl 3 o .~ t t o o~ t'l ~; _ tJ 3 tl3 ~_ O O O
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~

--t~ Cll t=~ ~tl~ O
3 ~ 2 t t ~ F

SU3STIT'JTE S11EET IRULE 261

Claims (26)

1. A method for monitoring performance of an incubator module said incubator module being suitable for incubating multiple samples simultaneously at a defined temperature (e.g. for incubating microtitre plates). said incubator module being comprised in an automated system for assaying multiple samples optionally in multiple simultaneous assays e.g. in multiple microtitre plates), said method comprising 1) measuring the absorbance of a temperature dependent solution in a photometer (phot 2) at at least two different wavelengths .lambda.1 and .lambda.2, wherein the absorbance of the temperature 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 temperature dependent solution comprising a buffer with a temperature dependent pH and an acid-base indicator with an absorbance spectrum linked to pH and said wavelengths being selected such that .delta..lambda./.delta.T is positive for one wavelength and negative for the other, said measuring providing absorbance values Atest.phot 2,.lambda.1 and .lambda.test,phot2,.lambda.2, wherein .lambda.test.phot221 indicates the absorbance (A) determined for the temperature dependent solution (test), using the photometer (phot 2) at wavelength .lambda.1 and Atest,phot indicates the absorbance (A) determined for the temperature dependent solution (test), using the photometer (phot 2) at wavelength .lambda.2, 2) saving data of step 1)in a data file 3 ) measuring the absorhance of a temperature independent calibration solution for the two wavelengths .lambda.1 and .lambda.2 of step 1) in the photometer (phot 2), said temperature independent calibration solution exhibiting the same absorbance spectrum as the temperature dependent solution at the defined temperature, said measuring providing absorbance values Acal.phot 221 and Acal.phot 222 wherein Acal.phot indicates the absorbance (A) determined for the temperature independent calibration solution (cal), using the photometer (phot 2) at wavelength .lambda.1 and Acal.phot 222 indicates the absorbance (A) determined for the temperature independent calibration solution (cal), using the photometer (phot 2) at wavelength .lambda.2 and 4) saving the data of step 3) in a data file 5) calculating the actual temperature T of the temperature dependent solution using the saved data and the formula In (Atest,phot2..lambda.1/Atest,phot2..lambda..2)=.alpha."+.beta.'.T, wherein .alpha."=.alpha.'- In (Acal,phot1..lambda.1/Acal,phot1..lambda.2) + In (Acal,phot2..lambda..1/Acal,phot 2..lambda.2) with the values of .alpha.' - In (Acal,phot1..lambda./Acal,phot1,.lambda.2) being either predetermined or else being obtainable by subjecting the temperature independedent calibration solution to absorbance measurement on a further photometer (phot1) for the two wavelenghths .lambda.1 and .lambda.2 of step 1) thereby obtaining Acal,phot 1..lambda.1 and Acal,phot 1..lambda.2. wherein Acal,phot 1..lambda.1 indicates the absorbance (A) determined for the temperature independent calibration solution (cal), using the further photometer (phot 1) at wavelength .lambda.1 and Acal,phot 1..lambda.2 indicates the absorbance (A) determined for the temperature independent calibration solution (cal), using the further photometer (phot 1) at wavelength .lambda.2 and saving the data of step 5 in a data file and subjecting the temperature dependent solution to absorbance and temperature measurement on the further photometer (phot 1) for the two different wavelengths .lambda.1 and .lambda.2 of step 1) thereby obtaining absorbance values Atest,phot 1..lambda.1 and Atest,phot 1..lambda.2. at known temperatures from which .alpha.' and .beta.' can be calculated usintg the formula In (A.lambda.1/A.lambda.2) = .alpha.'+ .beta.'.T and saving the data in a data file6) comparing the amended temperature or amended temperature distribution of a temperature dependent solution incubated in the module with the defined temperature the module is set to ascertain.

2. A method according to claim 1 wherein calculating means are used to correct the absorbance determined in the photometer (phot 2) with regard to the cooling down effect that occurs between the locations of incubation and absorbance measurement

3. A method according to claim 1 or 2 wherein a first microplate filled witll temperature independent calibration solution is transported to the photometer (phot 2) and the absorbance is read at the two wavelengths .lambda.1 and .lambda.2, measurement data is saved in a data file and the microplate is tahen 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 module and after completion of incubation transported to the photometer (phot 2), where the absorbance is read a number of times at the two wavelengths .lambda.1 and .lambda.2 at time intervals preferably controlled by a software timer and all data is saved in a data file.

4. A method according to claim 3, wherein the time interval between incubation termination and the first reading in the photometer (phot 2) is as short as possible and is limited by the time required for transport of the microplate from incubator module to the photometer (phot 2), said the interval preferably being less than 45 seconds, suitably being 25-35 seconds.

5. A method according to claim 3 or 4 wherein the time interval between absorbance measurements in the photometer (phot 2) is determined by the length of time required for the measurement of the absorbance at the two wavelengths .lambda.1 and .lambda.2 and the length of time required to save the data in a file, said time interval in general being longer than 25 seconds, preferably being less than 45 seconds and suitably being 30 seconds.

6. A method according to any of the preceding claims, wherein the temperature determined on the basis of absorbance data from the photometer (phot 2) is corrected for the cooling down that occurs between transport from incubator module to said photometer using calculating means on the basis of a mathematical function fitting the pattern of cooling down such that the regression coefficient R2 is equal to or larger than 0.98.

7. A method according to any of the preceding claims, wherein calculating means are employed enabling a function for absorbance against time to be fitted in the least squares sense on the absorbance measurement data obtained in the photometer (phot 2) enabling calculation of actual absorbance in the incubator module at the moment of taking the temperature dependent solution from the incubator module i.e. at time zero thereby enabling subsequent calculation of the actual temperature in the incubator module at time zero.

8. A method according to claim 7 wherein the function is an nth order, suitably a second order polynomial for the absorbance to the relation like A(t)= a + b t + c . t2, with a, b and c being constants and a being the absorbance at t=0 i.e. at the moment the temperature dependent solution is removed from the incubator module to be transported to thephotometer (phot 2).

9. A method according to any of the preceding claims, wherein calculating means are also used for filtering out any high frequency components in the temperature distribution due to imprecision of the photometer (phot 2).

10. A method according to claim 9, wherein the filtering out occurs using a two dimensional low pass filter.

11. A method according to any of the preceding claims, wherein the effect of interaction between the multiple samples of e.g. a microtitre plate is taken into account in the calculation of the temperature in the incubator module by fitting a two dimensional linear model in the sense of the least squares to the temperature data of a part of a matrix of samples i.e. two dimensional linear regression e.g. in a square block of nine wells for a microtitre plate.

12. A method according to claim 11 wherein the calculation of the temperature in the incubator module by fitting a linear model in the sense of the least squares to the temperature data of a square block of nine wells i.e. two dimensional linear regression e.g.
for a microtitre plate involves expressing the temperature after filtering as e.g.

TfiltD6 = f1. TC5 + f2. TC6 + f3. TC7 + f4. TD5 + f5. TD6 +
+ f6. TD7 + F7. TE5 + f8. TE6 + f9. TE7 (8) wherein C.D and E represent the row denomination of the square block and 5, 6 and 7 represent the column denomination of the square block, with f1 through f9 being constants which can be calculated.

13. A method according to claim 12, wherein use is made in the calculation of Model Tfilt= a.rownumber + b.columnnumber + c (a, b and c are constants) and minimising in the sense of the least squares results in function F;

F = .SIGMA.(Tfilt(welli)- T(welli))2 (i=1..9) (10) with the following being valid:
- for a well not at the edge of the microplate fi=1/9 - for a well at the edge of the microplate and not at a corner f1=f5=f6=2/18, f7=f8=f9=-1/18 with TfiltA6 = f1. TA5 + f2. TA6 + f3 TA7 +f4. TB5 +f5. TB6 +
+ f6. TB7 +f7. TC5 +f8. TC6 + f9. TC7 (11) - for a well at the corner f1=8/18, f2=f3=5/18,f4=f5=f6=2/18,f7=-1/18,f9=-4/18 as in TfiltA1 = f1.TA1 + f2.TA2 +f3.TB1 + f4.TA3 + f5TB2 +
+ f6.TC1 + f7.TB3 + f8. TC2 + f9.TC3 (12)

14. A method according to any of the preceding claims, wherein the buffer system of the temperature dependent solution has a large .delta.pH/.delta.T preferably at least an absolute value of 0,02 pH units/°C.

15. A method according to any of the preceding claims wherein the temperature dependent solution exhibits large .delta.A/.delta.pH vahles, with large being sufficient to result in at least a precision of 0,1°C and an accuracy of at least 0,5°C, preferably 0,3°C.

16. A method according to any of the preceding claims, wherein the solubility of the indicator in the temperature dependent solution and the temperature independent calibration solution is sufficient to ensure measurable and reliable absorbance values, preferably absorbance values between 0,5-1,5 AU at the defined temperature and wavelengths .lambda.1 and .lambda.2.

17. A method according to any of the preceding claims, wherein the concentration of acid-base indicator in the temperature dependent solution and the temperature independent calibration solutions to be used in photometer (phot 1) and photometer (phot 2) is such that the ratio of the length of the lightpaths through the samples in the two photometers is identical to the ratio of the concentrations.

18. A kit suitable for carrying out a method according to any of the preceding claims comprising at least a container comprising a temperature dependent solution exhibiting at least an absorbance higher than 0,5 AU at wavelengths .lambda.1 and .lambda.2 at the defined temperature, said temperature dependent solution comprising a buffer system with a temperature dependent pH and an acid-base indicator with an absorbance spectrum linked to pH and a container comprising at least one temperature independent calibration solution exhibiting an absorbance spectrum identical to that of the temperature dependent solution at the defined temperature, said temperature independent calibration solution preferably comprising a composition as close to that of the temperature dependent solution as possible.

19. A kit according to claim 18, wherein the pH of the solutions is a) in the working range of the buffer system of the temperature dependent solution at the defined temperature at which measurement is to take place, said defined temperature being within a desired temperature range, preferably being within the range 20-60°C, b) in the indication range of the acid-base indicator at the defined temperature at which measurement is to take place, c) such that the absorbances at the two wavelengths .lambda.1 and .lambda.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 CO2 absorption.

20. A kit according to claim 19 comprising multiple temperature independent calibration solutions for a number of defined temperatures, wherein the pH of each temperature independent calibration solution is selected such that the absorbance spectrum of the temperature independent solution is equal to the absorbance spectrum of the temperature dependent solution at the defined temperature.

21. A kit according to any of claims 18-20, wherein the temperature dependent buffer comprises an aqueous solution of citrate, tartrate, phtalate, phosphate, Tris(hydroxymethyl)aminomethane, Borax or sodium bicarbonate preferably of Tris(hydroxymethyl)aminomethane.

22. A kit according to any of claims 18-21, wherein the acid base indicator is Cresol Red and is present in soluble form.

23. A method according to any of claims 18-22, wherein the temperature independent calibration solution comprises HEPES, phosphate and a Cresol Red solution and ispreferably further identical to the temperature dependent solution which comprises Tris(hydroxymethyl)aminomethane as buffer and a Cresol Red solution as acid-baseindicator.

24. A kit according to any of the claims 18-23, wherein said containers further comprise agents against microbial degradation such as azide and preferably cinnamaldehyde and gentamicin sulphate.

25. Use of a method or a kit according to any of the preceding claims for determining the temperature dispersion of the incubator module at a number of different defined temperatures, said method or kit requiring a number of temperature independent calibration solutions equivalent to the number of different temperatures.

26. Use of a method or a kit according to any of claims 1-24 for determining the temperature dispersion of the incubator module in an automated multi-sample system whilst the system is in normal operation.