effect and factors on the regulation temperature IFCC Document

Journal of Automatic Chemistry of Clinical Laboratory Automation, Vol. 9, No. 2 (April-June 1987), pp. 82-86

The effect of instrumental andenvironmental factors on the thermalregulation of the temperature ofincubation IFCC Document (1986)

Prepared for, and on behalf of, the International Federation ofClinical Chemistry’s Expert Panel on Instrumentation by:

C. A. BurtisChemical Technology Division, Oak Ridge National Laboratory, PO Box X,Bldg. 4500N, Oak Ridge, Tennessee 37831-6194, USA

Preface

In 1982 the International Federation of ClinicalChemistry’s (IFCC), Expert Panel on Instrumentation,issued a statement recommending that 37C be thepreferred temperature of incubation for routine methodsin clinical chemistry.

Subsequently, the IFCC Scientific Committee formed ajoint working party, consisting ofDr R Rej, the Chairmanof the Expert Panel on Enzymes (EPE), and Dr C. A.Burtis, a member ofthe Expert Panel on Instrumentation(EPI), to prepare a report on incubation temperature.After initial discussions, it became apparent that basic-ally there were two fundamental aspects of the tempera-ture question: (1) the thermodynamic effect on theanalytical process in question; and (2) the thermalregulating capabilities of an instrument in the environ-ment in which it is expected to operate. With regard to thelatter, to achieve and maintain temperature regulation ina reaction cuvette, the opposing actions ofprocessing thatadd and remove heat to its contents must be balanced. Toachieve this balance within an acceptable time frame,heat is first generated and supplied by a regulated heatsource. Heat can then be removed either passively byallowing it to dissipate into the environment or activelyby some type of regulated device. Since the ambienttemperature ofthe environment in which an instrument isoperated has a direct and significant effect on theeffectiveness of passive cooling, regulated cooling isneeded when the temperature differential (AT) betweenthe ambient temperature and the set point temperature isless than 5 C. Since it is often difficult to maintain a ATof 5 at a set point of 30 C, 37 C was recommended bythe EPI in 1982. However, in some countries during thehot, summer seasons, the prerequisite AT of 5C isdifficult to maintain even at 37 C.

Expert Panel (Scientific Committee Analytical Section, IFCC)members are: T. D. Geary (Australia), Chairman, N. Alpert(USA), P. Bonini (Italy), C. A. Burtis (USA), and T. Sasaki(Japan).Comments on the document, including comments from theviewpoint of languages other than English, are encouraged andshould be sent to Dr Burtis at the Oak Ridge NationalLaboratory.

82

Since modern instruments are designed to operateworld-wide in a variety of environments, the EPI recom-mends thatfuture instruments be designed to provide both regulatedheating and cooling capabilities. Improved temperaturecontrol should result and the effect of ambient tempera-ture will be minimized.

The following document has been prepared in support ofthis recommendation by Dr Burtis on behalf of the EPI.

Introduction

To ensure comparable clinical results, considerable efforthas been, and will continue to be, expended on thedevelopment of a standardized measurement system foruse by clinical laboratories world-wide. As a consequenceof these standardization efforts, numerous referencematerials and methods are now available which havereceived consensus acceptance by a variety of nationaland international organizations.

One area of standardization which has proven to becontroversial and for which consensus has not beenobtained is that of selection of a single reaction tempera-ture at which all clinical analyses will be performed.During the past several years, various temperatures havebeen suggested and recommended [1-8]; however, thecontinuing debate has narrowed the choice down to either30 C or 37 C. Recently, on behalfofthe Expert Panels onEnzymes (EPE) and Instrumentation (EPI) of theInternational Federation of Clinical Chemistry (IFCC),an international survey on the topic of temperatureselection was conducted and the results published [9]. Aswould be expected from the history of the temperaturecontroversy, this document lists reasonable arguments forselection of either temperature. Clearly, the question oftemperature selection is a difficult problem and one forwhich an indisputable selection cannot be made.

There are two major components of the temperaturequestion, which can be categorized as analytical andinstrumental. Analytically, temperature is one of thevariables which an analyst can use to offset the kineticand steady-state thermodynamics of processes and phe-nomena such as reaction rates, antigen-antibody bindingand chemical complexes whose absorbance spectra istemperature dependent [10 and 11]. Instrumentally, theselection of a specific set point temperature as a set pointabout which the temperature of a reaction is to bemaintained has a direct effect on the design and cost ofan

C. A. Burtis/IFCC Thermal regulation of the temperature of incubation

analytical instrument and its ability to achieve andmaintain thermal regulation in the environment in whichit will be operated. The purpose of this document is todiscuss the various instrumental factors which influencethermal regulation and how they interact with those ofthe environment in which an instrument is to be operated.

Temperature regulation

The regulation ofthe temperature ofa liquid in a reactioncuvette at a specific set point is-a process in which the heatflux within the chamber is maintained and regulated for aspecific period of time (for example a steady-state isachieved). In practice, this entails the balancing of theopposing actions of heat input and heat loss via theseveral processes which add and remove heat to and fromthe system [12]. A block diagram of a generalizedtemperature control system is shown in figure 1.

process (Tm) to a controller which then regulatestemperature at a preset value (Tsp) while minimizingexcursions. Such a process is termed ’feedback control’.

Temperature measurement

The first step in the thermal regulation of a process is themeasurement of the temperature in the reaction cuvette.This measurement is made by some type ofa temperaturesensing device (usually a calibrated thermister, thermo-couple, thermometer, or thermochromic solution), whichis placed in or near to the reaction cuvette. Measurementsare generally based on calibrations which compensate foreffects resulting from the placement of the temperaturesensing device.

Heat addition

The function of any automatic control system is tomonitor a process variable (i.e. temperature of the liquid)and to alter controlled parameters (i.e. heat fluxes) via acontrol device to form a closed loop ofaction and responsewhich results in the desired system behavior 13]. In thegeneralized case shown in figure 1, there is a feedback ofinformation from the measured temperature of the

When the measured temperature ofthe reaction cuvette isless than the set point (Tm < Tsp) heat (q) is required andis added to the chamber until a thermal steady-state isobtained which results in Tm Tsp. The total heat addedis a combination of the heat generated from a regulatedheat source (active heating) and that supplied fromunregulated sources (passive heating).

ENVIRONMENTALSOURCES

PASSIVE PASSIVEHEATING COOLING

ACTIVEHEATING

(REGULATED) --- CUVETTE’--__== --_- ._=.--|-’MEASURED

1------:" liTEMPERATURESET-POINT/TEMPERATURE

i..T.,.mvl..< TS.p, _//OMPARE’__TM> TSp

INSTRUMENTALSOURCES

ACTIVECOOLING

(REGULATED)

TM TspSTEADY-STATE

Figure 1. Schematic representation ofa thermal regulation system and the effect ofenvironmental heat sources on it.

83


Regulated heat source

A regulated heat source actively generates a specificquantity of heat in a time frame consistent with theoverall time requirements of the system. In a simplecontrol system, the quantity ofheat provided is a functionof the temperature difference (AT) between Tm and Tspand the quantity of heat supplied from the unregulated,external sources. For a thermal regulating system to

function properly, the regulated heat source shouldprovide the predominant percentage of the total heatinput.

Unregulated heat sources

The ambient heat contained in the environment is one ofthe two major sources of passive or unregulated heat.When the ambient temperature approaches that ofthe setpoint and the resulting thermal differential becomes lessthan 5 C, the passive heat supplied from the environmentbecomes the major component ofthe total heat input and,since it is unregulated, control of the process is subse-quently lost.

The second source ofunregulated quantities ofheat is theheat produced from the routine operation of the system.When this system is a large, multicomponent device, thenconsiderable quantities of heat are generated during itsroutine operation. When the environment in which thesystem operates is controlled, then this endogenous heatcan be dissipated into the environment which functions asa high capacity heat sink. When the environment is notcontrolled and the ambient temperature approaches thatof the set point, then the heat capacity of this heat sinkbecomes limiting and this endogenous heat can become asignificant proportion of the total heat input. Thermalsteady-state is then difficult to maintain.

Heat transfer

As a specific quantity ofheat is generated, it is transferredto the reaction cuvette at a given rate. Its conduction is afunction of the cross-sectional area of the cuvette, thetemperature gradient across it, and the thermal conduc-tivity of the conducting medium [14]. There are threefundamental types of heat transfer: conduction, convec-tion, and radiation. All three may occur at the same timebut in any given system one is designed to predominate.Factors which influence the effectiveness of transfer byconduction are the heat conductivities and capacities ofthe materials used to fabricate the cuvette and also itscontents. In general, the heat transfer into and out of thecuvette will be faster through its walls than through itsliquid contents since the thermal conductivity of solids isapproximately 100 times greater than that ofliquids 14].

Heat loss

In order to maintain a constant heat flux through thereaction cuvette, its thermal regulating system must havethe capability not only to generate and supply heat butalso to remove excess heat. Heat can be actively removedby a regulated heat sink or passively lost throughunregulated heat sinks.

Regulated heat sinks

When Tm > rsp, a quantity of heat has to be removedfrom the reaction cuvette in order to achieve thermalsteady-state with Tm Tsp. With a regulated heat sink,specific quantities of heat can be removed at a constantand predictable rate. In general, a thermal regulationsystem that incorporates a regulated heat sink into itsdesign will cost more than those utilizing unregulatedheating sinks for heat removal. However, such a system iscapable of providing thermal regulation over a widerrange ofambient and set point temperatures than systemswhich use unregulated heat sinks for cooling purposes.

Unregulated heat sinks

Systems which utilize unregulated heat sinks are de-signed to lose heat by allowing it to passively dissipateinto the ambient environment. As a rule of thumb, it iscommonly assumed that for such systems to function, theambient temperature of the environment in which theyoperate has to be maintained (i.e. air-conditioned) atleast 5C below that of the set point temperature. Inaddition, cooling fans are also often included in suchsystems to hasten heat transfer and dissipation and toremove moisture, thereby minimizing condensation.

In an uncontrolled environment, the ambient tempera-ture can approach, and even exceed, the set pointtemperature. In this situation, passive dissipation of heatto the ambient environment does not occur.

Discussion

In the previous sections, the individual components thatconstitute or affect the thermal regulation on an instru-ment reaction cuvette(s) were discussed. Using thisinformation and the schematic shown in figure 1, thepertinent factors that will influence the selection of either30C or 37C as the set point temperature can beobjectively discussed.

Availability of an international practical temperature scalestandard

The accuracy of a temperature measurement depends onits calibration and traceability to a reference point on theInternational Practical Temperature Scale (IPTS).Reference points having relevance to the clinical labora-tory are the triple and boiling points of water. Recently,the melting points of gallium [15] at 29 772C andrubidium [16] at 3930C have been proposed asadditions to the IPTS and are available as certifiedreference materials from the National Bureau of Stan-dards (NBS).

The availability of a reference standard that is traceableto the IPTS has no direct effect on an instrument’sthermal regulating capabilities. However, a thermalregulating system which can be calibrated and traced toaccurate temperature measurements would ensure itscredibility and transferability between laboratories.Thus, any temperature selected as the set point should betraceable to the IPTS. The melting points of gallium andrubidium are close to the candidate temperatures of30 C

84


and 37C. Thus, a traceable and transferable accuracybase can be ensured for both temperatures.

Environmental conditions

The ambient environment in which it will be operated isone of the most important factors that is considered in thedesign and development of an analytical instrument. Inaddition, the interrelationship between the ambientenvironment and the thermal regulating capabilities ofaninstrument is a very important point to consider inselecting a set point temperature. As discussed earlier, thethermal regulation of a reaction cuvette and its contentsentails the dynamic balancing of the opposing actions ofheat input and loss. For this process to occur in apredictable and reproducible manner, heat fluxes mustbe controlled (figure 1) where possible and heat contribu-tions from unregulated sources must be minimized. Inpractice, this latter situation is ensured by controlling theambient temperature of the environment by air condi-tioning or localized cooling within the instrument. As arule of thumb, when the ambient temperature is main-tained at least 5C below that of the set point, theregulated heat source will provide most of the total heatinput. In addition, when the thermal differential (/ 7)between the ambient and set point temperatures is 5 C orgreater, than the ambient environment can also serve as alarge, high-capacity heat sink into which excess heat canbe transferred and passively dissipated. In fact, manyinstruments are purposely designed to use the ambientenvironment as the heat sink component of their thermalregulation system.

Thermal regulation systems begin to have problemsfunctioning when the ambient temperature begins toapproach that of the set point and the resultant thermaldifferential becomes less than the prerequisite 5C.Under this condition, the heat supplied from the unregu-lated source becomes the major component of the totalheat input and, since it is unregulated, control of theprocess is subsequently lost. In addition, transfer anddissipation of heat into the ambient environmentbecomes limiting thereby precluding its use as a heat sink.Obviously, under this condition, instruments which havebeen designed to utilize the ambient environment as a

component of their thermal regulation system would losecontrol of both the heat input and loss-regulatingfunctions of their system.

With a design specification that the AT between theambient and set point temperatures must be at least 5 Cor greater, then selection ofa specific temperature as a setpoint places certain requirements on the environment inwhich an instrument will be operated. For example, at aset point of 30C, the ambient temperature must bemaintained at 25C or less. Likewise, at 37C, theambient temperature should be less than 32C. In anair-conditioned laboratory, the ambient temperature ismaintained in a range of approximately 20-23 C. Obvi-ously, in such environments, either 30 or 37 C could beselected and an acceptable AT maintained. However,many laboratories are not air conditioned and when theyare located in areas that have warm to hot climates, thesummer temperature can easily range in the upper 20s to

low 30s. In fact, even air-conditioned laboratories locatedin such climates often have problems maintaining tem-

peratures in the low 20s. Consequently, thermal regula-tion is difficult when 30 C has been selected as the set

point since a AT of at least 5 C cannot be maintained.Even at a set point of 37C, problems may occur onextremely hot days.

To compensate for situations in which the ambienttemperature approaches that of the set point, certainoptions are available to an instrument designer. One, thelaboratory can be air-conditioned either centrallythroughout the entire building or by use of a portablewindow air-conditioning unit to cool an individual room.However, in some situations, neither is feasible. A secondoption is to include a regulated heat sink into the design ofthe instrument. This approach has been termed ’active’cooling and requires the addition ofa cooling unit into theinstrument and a means of removing moisture beforecondensation can occur. Examples of such units arecirculating water coolers, air coolers, air conditioners, ordevices based on the ’Peltier’ effect [17]. These latterdevices can also be used to supply as well as remove heat.

Mode ofoperationWhether an instrument operates in a manual or auto-matic mode has an impact on the selection on the set

point temperature. One of the initial steps in theoperation ofa thermal regulation system is to first achievethermal steady-state by adjusting heat fluxes so that thetemperature of the contents of the reaction cuvette ismaintained at the set point (Tin Tsp). Then, the systemmaintains temperature control by adding or removingheat as necessary.

In an automated instrument, the thermal regulationsystem can be designed to achieve and maintain a desiredtemperature in a time frame that is consistent with itsoverall design criteria. In a manual instrument, theproblem often encountered is the initial adjustment ofthetemperature of the reaction cuvette. Manual instrumentsoften utilize a thermal regulation system which can onlyadd heat with excess heat being removed only bypassive dissipation. In addition, the heat generatingcapacity and heat transfer characteristics of manualinstruments often result in thermally sluggish systems(i.e. there is a long time lag between a control action and a

system response). To compensate for such behaviorcharacteristics; the temperature of the cuvette and itscontents can be adjusted by a preheating step. The degreeof adjustment required depends on the ambient tem-

perature and the set point. If the ambient room tempera-ture is, for example, 22 , then the temperature of thecuvette has to be adjusted by either 8 or 15 , dependingon whether 30 C or 37 C is selected as the set point.

Usually, a preheating step is accomplished by placing thecuvette in an external thermostatted environment afterwhich it is then replaced into the instrument. Studieshave shown that even during the short time intervalbetween removing the cuvette from the thermostattedenvironment, drying it, if necessary, and placing it in thethermally controlled compartment of the instrument, a

85

Co A. Burtis/IFCC Thermal regulation of the temperature of incubation

drift in temperature occurs. The magnitude of the driftdepends on the difference between the ambient and setpoint temperatures; it is greater at set point 37 C than at30C. Consequently, manual instruments are oftenoperated at a set point temperature of 30 C. Since theIFCC enzyme reference methods have been developed bythe EPE using manual instruments, they have selected30 C as their recommended temperature.

Instrumental costs

A thermal regulating system (figure 1) involving regu-lated heating and cooling and strategically placed ther-mal sensors will provide the quickest and most tightly-controlled system to reach and maintain the temperaturearound a given set point. If properly designed, such asystem would provide acceptable performance indepen-dent of the ambient temperature and the temperatureselected as a set point. The primary disadvantage ofsuchan instrument is cost since it would be more expensive todesign and incorporate an active cooling component intoits thermal regulation system. However, the resultinginstrument would have the capability to operate inenvironments with a wider range of ambient tempera-tures irregardless of what specific temperature has beenselected as a set point.

References

1. BF.IOM.,,..I% H. U., Standardization of the reaction tem-perature for the determination of enzyme activity. Z. Klin.Chem. Klin. Biochem., 11 (1973), 39-45.

2. KIEBINO, R., HORB.R, M., G.RI-IARDT, W. et al., Recom-mended methods for the determination of four enzymes inblood. Scandinavian Journal of Clinical Laboratory Investigation,33 (1974), 291-306.

3. German Society of Clinical Chemistry, Recommendationsfor a uniform temperature for the estimation of enzymeactivities in clinical chemistry. Z. Klin. Chim. Klin. Biochem.,9 (1971), 464.

4. Socifit( Frangaise de Biologic Clinique, Standardisation dela temperature utilisee pour la measure des activitiesenzymatiques courantes en chimie clinique. Ann. Biol. Clin.,33 (1975), 51.

5. BowExs, G. N. Jr., B.MY, H. U., HOIBF.R, M. andMoss, D. W., IFCC methods for the measurement ofcatalytic concentration enzymes. Part 1. General considera-

tions concerning the determination of the catalytic concen-tration of an enzyme in the blood serum of man. ClinicaChimica Acta, 98 (1979), 163F-174F, and Journal of ClinicalChemistry and Biochemistry, 18 (1980), 89-95.

6. BRTAUBmRE, J. P., The optimal reaction temperature forenzyme activity measurements: 30 C, in Proceedings of theSecond International Symposium on Clinical Enzymology, editedby N. W. Tietz, A. Weinstock, and D. O. Rodgerson(AACC Press, Washington, D.C., 1976), pp. 303-309.

7. TIETZ, N. W., BATSAKIS, J., BAYSE, D. et al., Guidelines forphotometric instruments for measuring enzyme reactionrates. Clinical Chemistry, 23 (1977), 2160-2162.

8. STATLANB, B. E., Enzyme assay conditions: choice ofstandard reaction temperature for assaying activity valuesof enzyme in human sera (Document C-10, NationalCommittee for Clinical Laboratory Standards, Villanova,Pennsylvania, 1979).

9. HA.CKL, R., HORB.R, M. and Z.m.R, R., Report on thesurvey opinions concerning preferred incubation tempera-tures for measurement of enzymes (and possibly othercomponents) in clinical chemistry. Journal of ClinicalChemistry and Clinical Biochemistry, 20 (1982), 947-958; andClinica Chemica Acta, 129 (1.983), 245F-249F.

10. BURTIS, C. A., Sm.RT, L. E., BAIRB, M. A. and SAra’SON,E. J., Temperature dependence of the absorbance ofalkaline solutions of 4-nitrophenyl phosphate. A potentialsource of error in the measurement ofalkaline phosphataseactivity. Clinical Chemistry, 23 (1977), 1541-1547.

11. SPI.RTO, F. W., MACNIL, M. L. and BtmTIS, C. A., Theeffect of thermochromism on the measurement of cre-atinine. Clinical Biochemistry, 12 (1979), 18-24.

12. CHODOSH, D. F., LEVINSON, S. H., WEBER, J. L., KAMHOLZ,K. and BERKOFF, C. E., Automated chemical synthesis.Part 3: Temperature control systems. Journal of AutomaticChemistry, 5 (1983), 103-107.

13. COUGHANOWR, D. R. and KOPPEL, L. B. Process SystemsAnalysis and Control (McGraw-Hill Book Company, NewYork, (1965), pp. 101-110.

14. B.NNTT, C. O. and MYERS, J. E., Momentum, Heat and MassTransfer McGraw-Hill Book Company, Inc., New York,(1962), pp. 242-247.

15. MANaUM, B. W., The gallium meeting point standard. Itsrole in our temperature measurement system. ClinicalChemistry, 23 (1977), 711-718.

16. Standard Reference Material 1969: Rubidium Triple-PointStandard (National Bureau of Standards, Washington D.C.,1984).

17. HAaS.LAUd.R, U., ARNAUDOV, K. and FAUST, U., ZurProblematik des Temperierens in der Enzymanalytic, DT.Ges. f. Klin Chemie, 5 (1984).

FOOD ADDITIVES AND CONTAMINANTS

The latest issue of this international quarterly includes the following papers, which may be of interest to JACreaders:

Collaborative evaluation of a procedure for the determination of N-nitroso compounds as a group.Identification of substituted hydroxypyridines and hydroxypyrazines in caramel food colourings.Nickel in foods and the diet.

Sample copies from Taylor & Francis, Rankine Road, Basingstoke, Hampshire RG24 OPR, UK.

86

Submit your manuscripts athttp://www.hindawi.com

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation http://www.hindawi.com Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttp://www.hindawi.com

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation http://www.hindawi.com Volume 2014


CatalystsJournal of

Documents

effect and factors on the regulation temperature IFCC Document