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Analytical Letters
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Metrological Assurance of Biodevices: The New Method of Calibration of Biochemical Analyzers

Vladan Skerovic a; Veljko Zarubica a; Becko Kasalica b; Stevan Stojadinovic b; Ivan Belca b a Directorate of Measures and Precious Metals, Belgrade, Serbia b Faculty of Physics, Belgrade, Serbia Online Publication Date: 01 January 2009

To cite this Article Skerovic, Vladan, Zarubica, Veljko, Kasalica, Becko, Stojadinovic, Stevan and Belca, Ivan(2009)'Metrological

Assurance of Biodevices: The New Method of Calibration of Biochemical Analyzers',Analytical Letters,42:2,330 338
To link to this Article: DOI: 10.1080/00032710802507448 URL: http://dx.doi.org/10.1080/00032710802507448

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Analytical Letters, 42: 330338, 2009 Copyright # Taylor & Francis Group, LLC ISSN: 0003-2719 print=1532-236X online DOI: 10.1080/00032710802507448

SENSORS

Metrological Assurance of Biodevices: The New Method of Calibration of Biochemical Analyzers

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Vladan Skerovic,1 Veljko Zarubica,1 Becko Kasalica,2 Stevan Stojadinovic,2 and Ivan Belca2
1

Directorate of Measures and Precious Metals, Belgrade, Serbia 2 Faculty of Physics, Belgrade, Serbia

Abstract: Medical diagnostics and therapy use an extensive amount of electronics, optoelectronics, and laser devices. For their reliable functioning, calibration is unavoidable. Biochemical analyzers, which work on principles of absorptional spectrophotometry, became most widely present in medical biochemical laboratories. Construction of these devices is such, that in most cases, it does not allow calibration by usual methods, using neutral density (ND) filters as standards. For this reason, it was necessary to create a solution that could be used as a mean of comparison or even as a standard solution for methods of calibration of biochemical analyzers. In this article, the metrological characterization of solutions that are developed is presented, and the measuring uncertainty (ISO 1993) of methods used for calibration is estimated. The method that is developed provides metrological traceability to the primary spectrophotometric standard. With this method, metrological assurance in the field of the spectrophotometry in medical laboratories is completed. Keywords: Biochemical analyzers, calibration, measurement uncertainty, metrological, spectrophotometry, tracability

Received 3 September 2008; accepted 12 September 2008. Address correspondence to Vladan Skerovic, Directorate of Measures and Precious Metals, Faculty of physics, Belgrade. E-mail: vladanskerovic@dmdm.rs 330

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INTRODUCTION In modern medicine, a great variety of diagnostic techniques is used. Techniques have been developed for years using sensors and transducers to transform nonelectric quantities into electrical signals, which are further processed professionally by good measuring and signal processing devices. Therapeutical methods include also many electric therapies of alternating (ac) or direct (dc) currents. Future trends include, beside electromagnetic waves, beam techniques of acoustical and mechanical nature and associate measureing devices and electronics. Optical methods and transformations are also present with both coherent and incoherent sources and phenomena. Modern medical diagnostics is based on a number of optical instruments with corresponding electronics. Especially lasers have opened new fields in medical techniques. Absorption spectrophotometry, which uses Beer-Lamberts law for determining concentration, is widely used in medical and biological diagnostics. Calibration and testing of a wide range of spectrophotometric instruments is based on highly accurate measurement of spectral transmittance value, traceable to the national primary standard. According to measuring methods for traceability of spectral transmission values, calibration methods that are applied in spectrophotometry are divided into direct measurment methods and comparison methods. The direct measurement method is the basic method that is used for filter calibrations, that is, spectral transmission standards, which are used further for testing and calibration of spectrophotometers and photometers. Comparison methods assume that the filter, whether it is solid (glass) or liquid, is used only as a mean of comparison between a standard spectrophotometer and the device that is being calibrated. In that case, filter does not keep and reproduce unit value in a longer period of time, and it cannot be used as a standard. For calibration of biochemical analyzers where calibration can be done only by liquid samples, it is necessary to use proper solutions and to perform their metrological characterization, in order to define their usage, that is, their suitability to be declared as a mean of comparison or as standard. To do this, a number of solutions were made, from which those that were suggested to be the best after preliminary measurements were chosen. The idea was to make colored organic solutions, on a water basis, which significantly makes their further usage easier, because distilled water is used as a zero (referent) solution, and there is no staining (soiling) of devices flow-through system during the measurement.

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METROLOGICAL CHARACTERIZATION OF THE SOLUTIONS In this article, examination was performed on three different solutions of organic colors. At the beginning, all three solutions were symbolically marked as CR01, BY02, or LJ03. The spectral characteristic (an influence of radiation wavelength and spectral bandwidth), time stability of transmission value that they reproduce, and temperature influence were identified as basic metrological characteristics that are important for their classification.

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Spectral Characteristics of the Solutions On Figs. 13, spectral characteristics of examined solutions are presented. The solution CR01 was made in three different concentrations, that is, three levels of transmission. Considering that first idea was to use this solution as a mean of comparison, the data of precise concentration is not necessary. The other two solutions were made in only one concentration each for this examination, of nominal value of transmission around 50% at 550 nm. All measurements were made in the wavelength range of 400 nm to 700 nm. It can be seen from Fig. 1 that solution CR01 has almost ideally flat spectral characteristic, that is, that it is almost completely spectrally neutral, which qualifies it, from this point of view, as suitable for all kinds of calibration in the wavelength range of interest. Solution BY02 presents suitable spectral characteristic in 400 nm to 530 nm range, whereas solution LJ03 (Fig. 3) presents relatively flat and smooth curve of spectral transmission from 400 nm to 640 nm, with

Figure 1. Solution CR01 in three different concentrations.

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Figure 2. Solution BY 02.

the flat spectral characteristic around 550 nm to 640 nm. It can be concluded that solution LJ03, on the basis of its spectral characteristic, is suitable for the majority of applications in range of 400 nm to 640 nm, and, in combination with solution BY02, it can be qualified as an excellent solution for practically all applications in a given wavelength range. Criteria for such analysis are influence of wavelength accuracy on measurement of transmission and influence of spectral bandwidth on measurement of transmission. Component of measurement uncertainty of spectrophotometric measurements, uk, that originates from wavelength error, is calculated

Figure 3. Solution LJ03.

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from the product of error of wavelength setting u1 and change of transmission with wavelength as uk u1 (Dsk=Dk) (Manoochehri and Ikonen 1995). During measurements with a standard spectrophotometer, with a wavelength accuracy is 0.3 nm, this component of uncertainty is reduced to a level of 105, which makes this influence negligible. In commercial biochemical analyzers, wavelength accuracy is declared to be 1 nm and 2 nm, which, due to previous analysis of measurement results, are expressed as sufficient accuracy to allow this influence to be neglected. A general rule that can be applied for the influence of spectral bandwidth is that the highest value of spectral line width, where measurements of transmission with measurement uncertainty not higher than 1% are possible, should be 1=20 of symmetrical natural line width, at the half of the intensity, at the wavelength where measurement is performed, of a material in which transmission is measured (NIST 1994).

Stability of the Spectral Transmission Value Measurement of stability of the spectral transmission value was performed at primary spectrophotometric system (Skerovic et al. 1998), for each of the examples, at a few characteristic wavelengths, in 5-day periods, 10 h every day, and the temperature in the laboratory was within 23 to 25 C. In Fig. 4, a result of monitoring of the stability of the solution LJ03 during 3 days is presented, at three different wavelengths (the conduct is the same at the other wavelengths, but because of different transmission values, they could not be presented in the same figure). Measurement of

Figure 4. Three-day stability of the solution LJ03.

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the solution BY02, which is of the same type, practically gave the same results (only with different transmission values). From the measurement results, 2-day and 3-day stability is calculated, where an average value of standard deviation at all wavelengths where the measurement was performed is taken as a measure. For daily stability of transmission value, the result was 0.00254, i.e., 0.25%. For 3-day stability, the result is the value of 0.00525, that is, 0.525%. A clear linear trend of decrease of transmission in time can be seen from Fig. 4, from which, in a simple manner, a linear extrapolation could be obtained. However, this was not done, considering that such a change of transmission of the solution is caused exclusively by its evaporation, so it is also in temperature function, which would make every extrapolation unreliable, because it would assume that solution has been always kept at the same temperature. On the basis of performed analysis of measurement results, it can be concluded that solutions BY02 and LJ03 can be used, with great reliability, as means of comparison in the calibration method. By solving the problem of evaporation, and, first of all, defining the proper way of keeping the solutions, the time of their stability and their behavior in predictable way can be significantly prolonged. Examining the solution CR01, results for daily stability (in 5-h intervals) of around 0.5% were obtained, and for each separate day, the same average dispersion was obtained. On the other side, longer stability, including from day to day stability, always was higher than 1%, and there was no clear linear trend as in the case of the BY02 and LJ03 solutions. The reason for this is the precipitation of the solution CR01, which is identified during the testing. Considering this, it can be concluded that this solution can be used as a comparison mean only in a short time interval, when comparison is performed directly in the laboratory. On the other hand, this solution is especially suitable (considering its spectral characteristic) for calibration of devices, among which are biochemical colorimeters that have spectral bandwidths of around 50 nm.

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Temperature Influence Temperature influence on the transmission characteristic of the examined solution is determined by measurement of the solution LJ03, on temperatures of 23 and 26 C. An average change of transmission of the solution of less than 0.2% is obtained by measuring at a few characteristic wavelengths. Because the temperature influence of 0.2% is in the range of obtained dispersion of measurement results of solution transmission in the environmental conditions of 23 2 C, correction for temperature

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influence (which could be determined) was not performed. [AT A22 (1 CA(T22)) (NIST 1994), where are AT is absorbance at a given temperature, A22 is absorbance at 22 C, and CA is the experimentally obtained coefficient.] MEASUREMENT UNCERTAINTY Overall measurement uncertainty of the calibration of biochemical analyzers by method of comparison using colored organic solutions as means of comparison is evaluated from measurement uncertainty of the primary spectrophotometric system (Skerovic et al. 2008), reproducibility of measurements (which is expressed as stability of the spectral transmission value of the reference solutions), and uncertainty components resulting from influential parameters (bandwidth and temperature). All the measurements, including those for metrological characterization of the solutions and calibration measurements of the solutions in comparison methods, are performed at primary spectrophotometric system (Fig. 5, Skerovic et al. 2008). Measurement uncertainties budgeted for the primary spectrophotometric system, given in Table 1, are average values for wavelength range of 500 nm to 1000 nm, for 4-nm bandwidth measurements for the filter of nominal transmittance of 10%. The numbers in the table are relative values. The obtained value for nonstability of transmission values that the solution reproduces over time and under conditions in which calibration by a comparison method is performed is 0.254%, and in combination with measuring uncertainty of primary standard spectrophotometric system (in a given range, NIST 1994), which is 0.0856%, give a combined

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Figure 5. Scheme of spectrophotometric system.

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Table 1. Uncertainty budget Parameter Repeatabilty Nonlinearity Wavelength Setting Beam displacement Interflection Obliquity effect Polarization Drift RMS total Type A 0.000603 0.0005 0.1 nm 0.00004 Corrected Type B Uncertainty in special transmittance 0.000603 0.00045 0.00003 0.00004 0.00006 0.0004 0.000856

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measurement uncertainty of the method of 0.286% (k 1), that is, 0.536% (k 2). CONCLUSION It can be concluded that, on the basis of certain spectral characteristics, a solution suitable for practical calibration can be chosen, depending on the type of the device that is calibrated and the spectral range of the calibration. This confirms and guarantees that calibration of biochemical analyzers can be performed, in any case and under different conditions, with measurement uncertainty always under 1%, which is sufficient to calibrate the majority biochemical analyzers (OIML 2004). The method that is developed provides metrological traceability to a primary spectrophotometric standard. With this method, metrological assurance in the field of the spectrophotometry in medical laboratories is completed. REFERENCES
Guide to the Expression of Uncertainty in Measurements. First ed., 1993. International Organization for Standardization, Geneva, Switzerland. Manoochehri, F. and Ikonen, E. 1995. High-accuracy spectrometer for measurement of regular spectral transmittance. Applied Optics, 34: 36863692. NIST. 1994. Glass Filters as a Standard Reference Material for SpectrophotometrySelection, Preparation, Certification, and Use of SRM 930 and SRM 1930 (Special Publication 2260-166). NIST. US Government Printing Office, Washington, DC.

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Skerovic, V., Vukadin, P., Zarubica, V., and Zekovi, L. 2008. Realization of primary spectrophotometric system. Measurement Science and Technology, 19 085103 (6 pp.). DOI: 10.1088/0957-0233/19/8/085103. OIML. 2004. R 135: Spectrophotometers for Medical Laboratories. International Recommendation. Bureau International de Metrologie Legale, Paris, France.

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