Trends in Analytical Chemistry, Vol. 23, No. 5, 2004 Trends

Current practice in the assessmentand control of measurementuncertainty in bio-analyticalchemistry

Malcolm Burns

There is increasing recognition that estimation of measurement uncertainty

is valuable, yet very few areas implement a full measurement-uncertainty

budget with results. This article examines the advantages and the disadvan-

tages of uncertainty estimation, and illustrates some of its current uses and

problems within bio-analytical chemistry.

ª 2004 Published by Elsevier B.V.

Keywords: Analytical measurements; Measurement uncertainty; Quality control

*Tel.: +44-(0)208-943-7444;

Fax: +44-(0)208-943-2767;

E-mail: Malcolm.Burns@

lgc.co.uk

Malcolm Burns*

BioAnalytical Innovation Team,

LGC, Queens Road,

Teddington,

Middlesex TW11 OLY,

UK

0165-9936/$ - see front matter ª 200

1. Introduction

For a given analytical assay, uncertaintymeasurements attempt to identify allcomponents of variability and makereasonable estimates of their effects uponthe end result. As such, measurementuncertainty is a critical aspect in manyscientific disciplines, not least of which isbio-analytical chemistry.

Measurement uncertainty is closelylinked to quality control (QC) and qualityassurance (QA) aspects involved in meth-od validation. ISO 17025 [1] centres onclient interactions, method validation andtraceability of materials. As the estimationof measurement uncertainty is a criticalelement in many quality procedures for alaboratory, it is included under the ISO17025 heading [2,3].

Laboratories are gradually becomingmore aware of the need for, and benefit of,submitting full uncertainty estimates re-garding their analytical results, and the ISO/IEC Guide to the Expression of Uncertaintyin Measurement (GUM) [4] is increasinglybeing seen as a benchmarking publicationin this respect [5]. Publications are also

4 Published by Elsevier B.V. doi:10.1016/S0165-9936(04)00523-0

available detailing how these concepts canbe applied in chemical measurement [6].

However, whilst it is acknowledged thatthe principles outlined in the GUM arebeneficial to an analytical laboratory andthere is universal recognition behind thebenefits of evaluating measurement uncer-tainty acrossmany scientific disciplines, theimplementation of these guidelines for esti-mating measurement uncertainty is diffi-cult. The application of the principles oftenrequires expert knowledge of the scientificsubject field itself, and specialist guidelinesmay need to be written according to whichparticular assay or field within bio-analyti-cal chemistry is being assessed [3].

2. Implementation

As the issues regarding the implementa-tion of a successful measurement-uncer-tainty estimation are of fundamentalimportance to any laboratory that wishesto conform to ISO standards and produceresults of the best quality, many areaswithin bio-analytical chemistry have im-plemented their own uncertainty budgets.The examples that follow serve to illus-trate the diversity of sectors within bio-analytical chemistry that are attemptingto implement uncertainty estimation intheir work. The list also highlights thedifficulties that are sometimes encounteredin trying to use measurement-uncertaintyestimates, and shows that implementationof measurement uncertainty must beconsidered on a subject-by-subject basis.

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2.1. Method validation and QCWithin this sector, measurement uncertainty has itsroots in the traceability of chemical standards, and, assuch, its principles are applied in areas such as evalua-tion of reference materials, and to broad ranging issues,such as the applicability of equipment and assays.

Many analytical procedures use calibration curves orreference materials to produce a result. As such, one ofthe most fundamental evaluations of measurement un-certainty comes from addressing uncertainty associatedwith the values of these reference materials, and this hasbeen attempted in some detail [7]. A further publicationdetails a laboratory-capability survey, where uncertaintyestimates were used in assessing the suitability offorensic standards for use in routine measurements forprosecution purposes [8].

Regarding the analysis of sulphides in wine, a recentpaper [9] further outlines the application of measure-ment-uncertainty estimates in order to assess the per-formance of this analytical technique and show its‘‘fitness for purpose’’ in terms of material recovery. Amethod was evaluated by applying measurement un-certainty to a data set involving the use of spiked samples,and regression methods were used to assess the truenessof the analytical procedures. The technique is used toassess the trueness of samples that contain native analytespiked at several levels of concentration, when no blanksare available. The conclusion of this study was that someanalytical procedures can have incidental effects on theestimation of measurement uncertainty, and large biasassociated with the recovery of a technique caused theassociated uncertainty to be consistently underestimated.

In another reported example, a model was used tocorrelate historical proficiency-test data as the log of theinter-laboratory standard deviations versus the log of theanalyte concentration, independent of analyte or matrix[10]. The validity of this technique was shown for settingthe internal measurement quality objectives of a labo-ratory, and utilised the uncertainty budget of the labo-ratory to assign the maximum allowable variation ineach major step in the bias-free measurement system.

Estimation of measurement uncertainty allows theperformance of an assay to be assessed, and to be eval-uated in terms of the ‘‘fitness for purpose’’ of a method. Inthis respect, a new mass-spectrometry technique for thedetermination of iron isotopes in materials was shown tobe operating within specific performance criteria [11].

Measurements made during method validation canfrequently be used to construct uncertainty estimates;this has been detailed in the EURACHEM/CITAC Guideto Quantifying Uncertainty in Analytical Measurement[6].

2.2. Commercial markets and manufacturersThe authenticity of food products is a topic causingmuch current discussion. Measurement-uncertainty

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principles are applied to this sector of food analysis, andalso encompass other areas, such as evaluation of wines,determination of pesticide residues and increasing theefficiency of analysis with consequent cost-savingimplications.

For food analysis, an optimised uncertainty approachhas been suggested, taking into account uncertainty esti-mates from not only traditional areas, such as sampling,but also financial costs arising from misclassification ofmaterial. The implementation of this optimised uncer-tainty approach has reduced financial losses by anaverage of 65% in some areas of food authenticity [12].

Results from 61 separate inter-laboratory tests forpesticide residues on food products were examined in afurther study using measurement uncertainty [13]. Theaim was to determine whether or not a concentration-dependent relationship would hold for the muchnarrower range of chemicals and concentrations coveredin routine pesticide residue analysis.

2.3. Government agencies and regulatory bodiesMeasurement-uncertainty principles have been appliedto the field of forensics and regulatory legislation. TheCo-operation on International Traceability in AnalyticalChemistry initiated a study to assess the comparability offorensic ethanol standards used in different countries in1996. Measurement-uncertainty estimates were madeand the results discussed in the context of performancerequired to support routine measurement for prosecutionpurposes [8].

Within the US, cell-line K562 is the forensic controlmaterial for RFLP-DNA profiling. Over an eight-yearperiod in the 1990s, 51 proficiency tests were conductedon RFLP-measurement performance; they showed thatthe average measured size of some loci varied slightlyover time. These changes had an important effect uponthe overall inter-laboratory measurement uncertaintywith implications for use of the material [7].

2.4. Risk assessmentsEnvironmental quality issues can be addressed by riskassessment, and measurement uncertainty has beenapplied in areas such as wastewater treatment, airquality, impact of pesticides, analysis of organic pollu-tants and the design of equipment to specified criteria.

Regarding environmental management programs,measurement-uncertainty estimates have been imple-mented to predict the costs of construction of wastewatertreatment plants. These estimates should allow moreaccurate assessment of the economic feasibility ofnumerous water-pollution programs in Asia [14]. Re-lated to this, Monte-Carlo simulations have been used toevaluate measurement-uncertainty issues regardingriver water quality, and the results have helped in thedevelopment of control strategies for wastewatertreatment plants [15]. A further paper describes the

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application of measurement-uncertainty principles in theevaluation of triazines in groundwater from validationdata [5].

Gas chromatography-inductively coupled plasmamass spectrometry (GC-ICP-MS) was used in the detec-tion of butyltin compounds in sediment using a species-specific isotope dilution [16]. This study showed that themain contributor to measurement uncertainty withinthe assay was associated with the initial extraction fromthe sediment.

The application of measurement-uncertainty princi-ples to contaminated land investigations has beendescribed [17]. The continued accumulation of anychemical in the environment can have detrimentaleffects, and a further recent paper studies the evaluationof measurement uncertainty associated with measuredbio-accumulation factors in fish [18]. The study wasdesigned to identify what sampling procedures wouldlead to the least amount of variability, and showed thatfactors, such as the temporal variability of chemicalconcentrations in the water column, have a very greatinfluence.

Another recent paper focused on the measurementuncertainties involved in assessing risks to humanhealth from the exposure to benzene in the environment[19]. Whereas the main focus of the study was to eval-uate how well uncertainty judgements from expertswithin the field of benzene exposure correlated with theactual calculations, the paper provided evidence toillustrate the importance of uncertainty estimates in thefield of environmental monitoring.

Companies that produce commercial equipment areincreasingly recognising the need to evaluate measure-ment uncertainty in the systems they produce. Uncer-tainty principles have been applied in order tomanufacture fluid-flow-calibration systems and ambientair-sampling instruments of the highest quality [20].

2.5. Clinical chemistryActivities within the area of clinical chemistry wheremeasurement-uncertainty principles have been appliedinclude laboratory work, patient welfare and analysis ofblood-serum samples.

General guidelines on how to implement themeasurement-uncertainty issues outlined in the GUMbook with particular regard to clinical laboratory workhave been published [21]. Again, this highlights that theimplementation of the measurement-uncertaintyprinciples for a particular area requires specialisedknowledge. Each scientific discipline has to be consideredseparately, as it may produce unique problems to beovercome in terms of uncertainty assessment.

Measurement-uncertainty principles have been usedin order to estimate the amount of calcium and glucosefound in blood-serum samples [22]. Using the principlesoutlined in GUM, uncertainty components were quanti-

fied from the measuring system, calibration certificates,instrument specifications and literature. To facilitate thecalculations, a software package was used. This paperdescribed how the systematic procedure adopted can beeasily applied to many other measurands in clinicalchemistry. The work also emphasised that internalQC can provide much of the information needed inuncertainty evaluation.

2.6. Radiation monitoringMeasurement-uncertainty calculations have been ap-plied across many diverse fields, including radiationmonitoring. Following the Chernobyl reactor accident,studies through a European project in 1992–1993showed that re-suspended radioactive aerosols were stillpresent in the Chernobyl area. A potentially significantdose may result from inhalation and secondary con-tamination caused by re-suspended radionuclides, whichtherefore needed to be monitored. Discrepancies betweensampling units were found, and measurement-uncer-tainty estimation showed that most of this variation wascaused by an uncalibrated analytical system [23]. Theimplications for producing the best quality results withtheir associated measurement-uncertainty estimates onhealth and safety were critical in this area.

2.7. Health, fitness and sports sciencesUnder this rather broad subject heading, studies includethe application of measurement-uncertainty principles inthe identification and analysis of fat-soluble vitamins,drugs, and cytokines, and the impact of these moleculesupon human health.

Concentrations of fat-soluble vitamins were deter-mined from human sera in two epidemiological studies[24]. Intra-laboratory measurements of reproducibilitywere made, and the confidence in the results was shownto depend upon the distribution of the analyte within thepopulation, and also on the measurement uncertaintyinvolved.

Measurement-uncertainty principles have beenapplied to establishing the purity of organic certifiedreference materials (CRMs) used for drug analysis insports [25]. The National Analytical Reference Labora-tory in Australia had synthesised and characterisednumerous anabolic steroid-marker metabolites andsteroid-conjugate pure-substance CRMs. Analysis wasconducted using GC with flame ionisation detection (FID)and high-performance liquid chromatography (HPLC).Measurement-uncertainty assessment was involved inthe evaluation of the identity and the purity of thesechemicals. Demonstrating the full traceability of thesesteroid materials should facilitate their use as CRMs forthe detection of drug abuse in sport.

Studies were conducted to evaluate the uncertainty ofenzyme-linked immunosorbent assays (ELISAs) for thedetermination of cytokines interleukin-4 (IL-4), IL-5,

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interferon-c (IFN-c) and tumor necrosis factor-a (TNF-a)[26]. A formal uncertainty-budget approach was appliedto each cytokine in the ELISA. The study established thatthe major contribution to uncertainty came from themass concentration of the International Standards of theWorld Health Organisation, because the uncertaintylinked with these was not provided in the accompanyingcertificate but was evaluated by other means. The largestsources of uncertainty were external to the laboratory,hence the conclusion that there were only limited pos-sibilities of improving the reliability of the results.

3. The demand

There is increasing awareness that the full evaluation ofan analytical result can be achieved only by the esti-mation of the measurement uncertainty associated withthat result [27]. There is growing pressure on all labo-ratories to conform to quality standards in expressingthis uncertainty, and to evaluate and to document boththe uncertainty and traceability of their analyticalresults [3,5,26]. Of particular importance is the role ofuncertainty in its evaluation of a result to demonstrateregulatory compliance [19].

Such is the demand and the recognition behind im-plementing measurement-uncertainty strategies in ana-lytical science that on-line resources are currentlyavailable to allow scientists access to some of themore specialised techniques involved (http://www.measurementuncertainty.org).

A number of instructional courses currently availableoffer an introduction to some of the principles ofmeasurement uncertainty, and formal BSc programmesinclude modules on the subject.

The issue of measurement uncertainty is currently ofwidespread interest and is the focus of many discussions,as evidenced by the numerous conferences and work-shops on the subject. The diversity of these workinggroups illustrates the different strategies of implementingestimates of uncertainty within the field of bio-analyticalchemistry [28].

Additionally, conferences have been run in order todiscuss some of the measurement-uncertainty issuesassociated with commercially available instrumentation[20].

The demand for the use of measurement-uncertaintyprinciples in analytical laboratories has been furtherhighlighted in another article [3], which presented arelatively simplistic and easily applied model on how toevaluate uncertainty in clinical chemistry.

Building upon the principles of measurement uncer-tainty outlined in the GUM book, further detailed liter-ature has been produced discussing the application ofmeasurement uncertainty in the fields of scientific

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metrology, industry, and trade and commerce, as well asintroducing more advanced statistical concepts [6,29].

4. The difficulties

Although the majority of areas involved in bio-analyticalchemistry recognise the importance of measurement-uncertainty estimates associated with their results, thereare many arguments against implementing such esti-mates. One such argument is that the priority of anyresult should be to meet customer requirements, and, ifthe customer does not need an estimate of the uncer-tainty associated with a result, then the resources can bebetter invested in other areas of the analysis [2].

Additionally, many analytical scientists believe thatthe correct evaluation of measurement uncertainty maybe unrealistic or impossible to achieve in practice [27].Although guidelines exist for the implementation ofuncertainty budgets, practical situations often dictatethat these are not feasible; each area must be assessed ona system-by-system basis [28]. However, it can also beargued that previously generated analytical data, forexample through formal method validation, can be usedto make measurement-uncertainty estimates [6,27].

In the area of clinical chemistry, it often appears thatthe effort required to estimate a full uncertainty budgetmay be so great that it will be difficult to bring intogeneral use regarding patients’ results [21].

Other problems in the implementation of measure-ment-uncertainty principles appear to arise in the in-trinsic contradictions of the term and how it is defined.There is universal difficulty in evaluating uncertainty inpractice, even though the value of reporting the estimateof measurement uncertainty is regarded as beingbeneficial. Suggestions have been put forward to parti-tion the method into more structured elements, such asestimating measurement uncertainty for sampling,calibration, and analysis [3,5], so that the wholeanalytical process is better understood. Recognising thata full measurement-uncertainty estimation may be quitecomplicated, one article suggested a simplified approach,utilising a method for estimating the worst casemeasurement uncertainty for analytical methods [30].This method for a ‘worst case scenario’ is claimed to beadequate for routine determinations of measurementuncertainty in any system.

5. Further methods

Because the calculation of measurement-uncertaintybudgets can be a laborious, iterative task, computerprograms have been implemented to estimate the un-certainties associated with complex situations. Softwareis also available to help evaluate measurement

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uncertainty as part of method validation; other areashave had specialised software written to assist in theevaluation of measurement uncertainty [22].

The implementation of Monte-Carlo re-samplingmodels to investigate the combined effects of uncertain-ties associated with cardiac output was shown to be avaluable tool for the analysis of errors in measurementmethods. This use of computer intensive Monte-Carlomodels appeared to be in widespread practice to helpestimate measurement uncertainty [15].

6. Summary

Within bio-analytical chemistry, there is increasingrecognition that measurement-uncertainty estimationcan add value to analytical measurement. Any resultfrom an analytical laboratory must be given with anassociated uncertainty estimate, if that result is to con-form to quality regulations. Measurement uncertainty isnow strongly associated with QC and QA aspects of alaboratory, as well as being an integral part of methodvalidation, certification, and accreditation. Manufactur-ers of scientific instrumentation are showing an in-creased interest in the subject, so that they can ensurethat their equipment meets specified performancecriteria.

Estimates of measurement uncertainty play a centralpart in the accurate analysis of results in many subjects,including environmental analysis, clinical chemistry,food authenticity, health and fitness, sport, radiationmonitoring, and many legislative areas, as evidenced bynumerous published articles and conferences.

In principle, measurement uncertainty can allow alaboratory to identify factors that contribute the mostvariation associated with a reported result, and thenpotentially reduce any financial loss by examining thisfactor in greater detail. Measurement-uncertaintyestimation helps qualify an assay or instrument as being‘‘fit for purpose’’ and show it to be operating withindefined performance criteria.

Measurement-uncertainty estimates have beenutilised as diagnostic tools in identifying where routinemeasurement problems occur, as well as being used toincrease the accuracy of results in feasibility studies andrisk assessments by identifying sources of potentialvariability. For regulatory legislation, estimates ofmeasurement uncertainty are critical so that a level ofconfidence can be attributed to a reported result.

However, although the majority of analytical areasrecognise these benefits of measurement-uncertaintyestimation, the implementation of these principles isnot widespread. Because each assay uses differentapproaches to produce a result, each application must betreated on a case-by-case basis. Coupled with the re-quirement for the specialist knowledge of the particular

assay is the prerequisite for expert knowledge of theprinciples behind measurement uncertainty. The criteriarequired to implement full measurement-uncertaintyestimation are therefore rarely met, and many scientistssee that the effort spent in trying to achieve this can bebetter re-invested within the laboratory, for example byproducing more results.

The complex nature of measurement uncertainty hasled to the development of a number of specialised soft-ware applications to help in its evaluation. To simplifythe mechanics behind implementing measurementuncertainty, other strategies have shown that data pre-viously generated from method validation may be usedin uncertainty estimation, or have suggested thatmeasurement uncertainty be structured into a moreordered, basic process. Despite these attempts at greaterunderstanding behind measurement uncertainty, veryfew disciplines within bio-analytical chemistry regularlyimplement a full measurement-uncertainty budgetassociated with their results.

Acknowledgements

This work was supported under contact with theDepartment of Trade and Industry (UK) as part ofthe National Measurement System Measurements forBiotechnology (MfB) Programme ‘‘Assessing andcontrolling uncertainty in biological analysis’’.

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