METHOD AND APPARATUS FOR EXAMINING FLUIDS OF BIOLOGICAL ORIGIN

Abstract: An extinction spectrum is approximated in a first wavelength range by a combination of a merely theoretical curve and the spectrum of the pure first substance in a first wavelength range, and this evaluation is repeated in a second wavelength range by approximating the measured spectrum (62) by a combination of a hypothetical curve, the spectrum (64) of the first component with the already determined concentration, and the spectrum (65) of the pure second component. The hypothetical curves are preferably straight lines which are defined by slope and ordinate section. In the praxis of the quality test of blood, bilirubin and hemoglobin may be quantitatively determined, whilst the background together with the lipid component can be qualitatively examined by means of the differential spectrum.

Description: METHOD AND APPARATUS FOR EXAMINING FLUIDS OF BIOLOGICAL ORIGIN The present invention relates to a method for examining a fluid according to the preamble of claim 1. It relates further to devices for executing the method. Clinical laboratory tests are routinely performed on the serum or plasma of whole blood. The tests commonly employ a series of reactions which terminate after the generation of chromophores which facilitate detection by spectroscopic measurements. The accuracy of most spectroscopic tests is affected to some extent by in vitro interferences. In vitro interferences arise from the fact that biochemical analysis are performed in the complex matrices that make up biological fluids, e.g. serum, plasma or urine. These fluids contain numerous compounds that either have chemical groups that can react with the test reagents or can have the physical or spectral properties of the target analyte. Further, the chemical composition of body fluids can vary with the nature and the extent of disease processes. In vitro interferences can be classified into two classes: spectral and chemical interference. The most commonly observed interferences are hemolysis, icteria, and lipemia. Some 30 % of samples obtained from clinic or hospitalized patients are he olyzed, icteric, or lipemic. Main reasons for hemolysis are unskilled blood taking or sample preparation, for icteria the jaunice disease, and for lipemia fat nutrition before blood taking. The goal of sample quality monitoring is the determination of the interfering substances hemoglobin, bilirubin, and lipid prior to conducting fully automated clinical laboratory tests in order to provide meaningful and accurate test results. If a sample is sufficiently contaminated with interference substances, the test may either not be conducted or the test result may be flagged to indicate that it is not reliable. Particularly, such a test is desirable in connection with the use of clinical-chemical analyzers which perform most of the analysis of a sample fully automatically and without taking into account any particular characteristics or properties of individual blood samples. U.S. Patent Specification US-4,263,512 describes a known method and device for semi-quantitative sample quality monitoring of hemoglobin and bilirubin using multiple wavelength measurements on diluted serum samples. This method has the following disadvantages: - it does not provide a quantitative determination of any interfering substances contained in the sample, and - it requires a special and specific conditioning of the sample. Alternative methods are chromatographic or clinical-chemical determination of the concentrations of interfering substances. The chromatographic measurement requires a long measurement time and delicate instrumentation, whereas the clinical-chemical determination is not suitable for reagentless measurement. Therefore, it is an aim of the present invention to provide a method for estimating rapidly the concentration of at least one interfering component in a fluid biological sample to be analyzed by an in vitro diagnostic test. The above aim is attained with a method according to the invention as defined by claim 1. The remaining claims define preferred embodiments and applications thereof and means for carrying out the method. In a preferred method according to the invention, the combination or superposition of the extinction spectrum of this first one of the components in a pure state and a function approximating the background extinction is fitted to the measured spectrum of the fluid to be analyzed in a wavelength range, where the component to be determined shows a significant or characteristic shape of its extinction curve. The function approximating the background extinction may e.g. be a straight line, and in this case, the wavelength range is preferably chosen where the expected background extinction spectrum is similar to a straight line . The invention shall be further explained in referring to exemplary embodiments with reference to the Figure: Fig. 1: Schematic representation of a photometric spectrum measurement arrangement. Fig. 2: Extinction spectra of pure interfering substances and a standard blood serum. Fig. 3: Normalized extinction spectra of real whole blood sera, bilirubin and hemoglobin contribution being subtracted, and a reference lipid solution sample. Fig. 4: Evaluation method for sample quality monitoring. Fig. 5: Experimentally measured extinction spectrum of a real whole blood serum and results of the evaluation method. Fig. 6: Measured extinction spectrum of a strongly hemolytic whole blood sample and respective extinction spectra obtained by the examination method. Fig. 7: As Fig. 6 for a strongly icteric whole blood sample. Figs. 8, 9: Optically determined hemoglobin (Fig. 8) and bilirubin (Fig. 9) concentrations versus added concentrations for 125 independent test samples. Figs. 10,11: Optically measured hemoglobin respectively bilirubin concentration values vs. clinical- chemically measured concentration values for independent real whole blood sera. Figs. 12-15: Optically measured hemoglobin and bilirubin concentrations of 92 real whole blood sera and the respective CV values obtained using a state-of-the-art spectrometer. Fig. 16, 17: Low-cost versus state-of-the-art spectroscopically measured (Fig. 16) hemoglobin and bilirubin (Fig. 17) concentrations of 92 real whole blood sera. Fig. 18: Schematic illustration of a dip probe. A method according to the invention for sample quality monitoring of blood serum or plasma by optical absorption spectroscopy in the visible and near IR range is described hereinafter. The target measuring ranges are 0.1 - 10 g/1 hemoglobin, 2 - 20 mg/dl (1 dl = 0.1 liter) bilirubin and 100 - 2000 mg/dl lipid with a measurement accuracy of 20 %. The evaluation performed by the method according to the present invention yields e.g. the content of the hemoglobin and bilirubin. A quantitative determination of the lipid concentration by optical absorption spectroscopy is not possible due to a lack of a reproducible relation between light-scattering and lipid concentration. Therefore, a differential extinction spectrum is obtained by subtracting the hemoglobin and bilirubin contributions from the extinction spectrum of the target sample. It contains the spectral contributions of the lipid and the matrix, e.g. the blood serum or plasma, which can then be investigated for spectral anomalies. The method has been experimentally tested using a series of 125 synthetic test samples and a series of 92 real blood sera. Accuracy and reproducibility of the technique versus the performance of the spectroscopic measurement device are described and commented hereinafter. A basic setup for optical absorption spectroscopy for sample quality monitoring is shown in Fig. 1. A light beam 1 emitted by a multiple optical wavelength light source 2 is collimated by a lens 3, which directs light of spectral intensity Io(λ) to a target sample 6. The optical path within the target sample is denoted by d. A lens 4 collects transmitted light of intensity I (λ) , which is then detected by a spectral wavelength analyzer which has an input 5. Optical absorption is commonly characterized by the extinction E (λ) , which is defined as T τk = - EW . ( i ) In the presence of J interfering substances in the target sample, e.g. hemoglobin, bilirubin and lipid (i. e., J=3) , the extinction E (λ) of the target sample can be described by the linear combination E(λ) = ∑K ( λ) C j + E λ) , (2) 7=ι q*ι where K-, and C 3 are the specific extinction coefficient and the concentration of interfering substance j (j = 1, 2, ..., J) , respectively. The dilution of the target sample is denoted by q d ιi, i. e. (original concentrations) : (sample concentrations) = (1 :q d n) . E g is the extinction characteristic of the matrix, e.g. blood serum or plasma. In Figure 2 the extinction coefficients K H of hemoglobin, K B of bilirubin, and K L of lipid (Intralipid 20% [Pharmacia, Sweden]) in the visible and near IR range are represented by lines 10, 11 and 12 respectively. The extinction spectrum E g of a standard blood serum (Control Serum N (human) [Hoffmann-La Roche, Switzerland]) is represented in Fig. 2 by dashed line 13. Within the scope of sample quality monitoring, a minimum number N mιn = 4 of statistically independent extinction values E(λ n ) (n = 1, ..., 4) should allow the determination of the four unknown parameters in Eq. (2), i.e. the concentrations of the interference substances hemoglobin (C H ) , bilirubin (C B ) and lipid (C L ) , and the extinction component (E g ) corresponding to the matrix part. More reproducible results are expected by least squares fitting the mathematical model of the extinction spectrum defined by Equation (2) to N > N mιn measured values E (λ n ) (n = 1, 2, ..., N) in order to obtain best estimates of the values of C H , C B and C L . However, it is observed that the specific extinction coefficient K L (λ) of lipid is not reproducible in real blood sera, and this is mainly due to the statistical distribution of the size of the scattering centers in the lipid. Moreover, as shown by Fig. 2, the monotonically decreasing extinction spectrum of lipid versus wavelength lacks typical (local) characteristics. Therefore, the latter spectrum cannot be distinguished from the component (E g ) of the extinction spectrum which corresponds to the matrix. Figure 3 shows extinction spectra 31 of real whole blood sera, from which the hemoglobin and bilirubin contributions have been subtracted. These differential spectra 31 therefore represent the sum of the spectral contributions of the lipid and the matrix. The shown extinction spectra are normalized to the extinction at λ = 700 nm. The solid line 30 represents the extinction spectrum of the reference solution of Intralipid, the broken lines 31 represent the extinction spectra of several samples of real whole blood sera after subtraction of the extinction contributions of hemoglobin and bilirubin. Hence quantitative determination of the concentrations of hemoglobin (C H ) , bilirubin (C B ) and lipid (C L ) does not appear to be possible by measuring the optical extinction spectrum of the target sample and fitting the model in Equation (2) to the measured values E (λ n ) . Therefore, according to the invention, a sequential determination of first the hemoglobin (C H ) and then the bilirubin (C B ) concentration is proposed in order to obtain a differential spectrum E dlff by subtracting the hemoglobin and bilirubin contributions from the measured extinction spectrum E (λ) . The differential spectrum E dlff thus represents the sum of the contributions of the lipid (E L ) and the matrix (E g ) to the extinction spectrum E (λ) of the target sample, and may additionally be investigated for spectral anomalies over the whole spectral range. The bloc diagram in Fig. 4 summarizes an example of a proposed measurement and evaluation method according to the invention for sample quality monitoring. This method is based on approximating the differential spectrum E dlff = E g + E L in a limited wavelength range λ r by a straight line. In a first step 32, the hemoglobin concentration is determined from a first measured extinction spectrum E x (λ) in a wavelength range λ rh ≡ [545, 575] nanometer. A set of extinction values comprising those of spectrum Ei (λ) is represented by block 35. As shown by Fig. 2, within the wavelength range λ rh ≡ [545, 575] nanometer hemoglobin has typical spectral characteristics and the bilirubin contribution to the extinction spectrum is quasi negligible (Fig. 2) . In a curve fitting step 37, the mathematical model of the extinction spectrum defined by Equations (3) and (4) hereinafter is fitted by a linear least squares algorithm to Ni spectroscopically measured values Ei (λ n ) (n = 1, 2, ..., Ni) in the range λ rh . In curve fitting step 37, the extinction spectrum Ei (λ n ) is approximated by E^λ) = E d ι(λ) + E H (λ) (3) where E H (λ) is the hemoglobin contribution and is defined by E H (λ) = K H (λ)-^-C H (4) and Edi linearly approximates the sum of the lipid and the matrix contributions, and E d ι is a function defined by Edi = a 0 h + aihλ. Note that the parameters a 0h and a ih have no physical significance. Fitting step 37 delivers the best estimate of the values of the hemoglobin concentration C H and a 0h , a ih . This set of values is represented by block 38. In a second step 33, the bilirubin concentration is determined from a second measured extinction spectrum E 2 (λ n ) in the wavelength range λ rb ≡ [480, 545] nanometer. A set of extinction values comprising those of spectrum E 2 (λ) is also represented by block 35. In a curve fitting step 41, the mathematical model of the extinction spectrum defined by Equations (5) and (6) hereinafter is fitted by a linear least squares algorithm to N 2 spectroscopically measured values E 2 (λ n ) (n = 1, 2, ..., N 2 ) in the wavelength range λ rb In curve fitting step 41, the extinction spectrum E 2 (λ n ) is approximated by Eι(λ) = E H (λ) + E d2 (λ) + E B (λ) (5) where E B (λ) = K B (λ) — C B (6) a ώl is the bilirubin contribution, E H is the previously determined hemoglobin contribution, and E d2 linearly approximates the sum of the lipid and the matrix contributions and E d2 is a function defined in particular by E d2 = 0 b + aι b λ, and in general by E dk (λ, aι, S k) with i ranging from 0 to at least 1. Note that the parameters a 0b and ai have no physical significance. Fitting step 41 delivers the best estimate of the bilirubin concentration C B and a 0 b, aι b . This set of values is represented by block 42. In a further method step 34, using Equations (2), (4) and (6), the differential spectrum E d ιf f , , is obtained by the equation E Aff (λ) = E(λ) - E H (λ) - E B (λ) , (7) The set of values of E dlff is represented by block 45. This set of values can then be investigated for spectral anomalies and lead to results represented by block 46 which can be in particular indicative of an anomalous lipid content of the target sample. The method according to the invention can be used to determine the concentration of k pure substances contained in a target sample. For that purpose, in more general terms the method according to the invention comprises: (a) measuring a first extinction spectrum Ei (λ) of a liquid sample in a first selected wavelength range λ= λι,ι to λι, n , and (b) fitting an approximated spectrum E ] _(λ) to said first measured extinction spectrum Ei (λ) , said approximated spectrum E^λ) being a combination of a predetermined approximation function Edi(λ,a 1# sι) for the background extinction, with i ranging from zero to at least one, and a predetermined extinction spectrum E S ι(C S ι,λ) of a first pure component of concentration C≤i of the components to be determined, said fitting being performed by varying said concentration Csi of said first interfering component and at least two of said coefficients a-, S ι, so that the deviation between said first measured extinction spectrum Ei (λ) and said approximated spectrum E 1 (λ) is minimized in order to determine the concentration of said first interfering component, and said first selected wavelength range being so selected that the concentration C S ι of said first interfering component can be determined unambiguously. In a preferred embodiment the approximated spectrum E ] _(λ) is the sum of said predetermined approximation function Edi(λ, a 1#S ι) for the background extinction, and said predetermined extinction spectrum E Sι (C S ι,λ) of said pure first component of concentration C S ι. In order to determine the concentration of a k pure component of a target sample a method according to the invention further comprises (a) measuring at least one further extinction spectrum E k (λ) of said liquid sample in at least one further selected wavelength range λ= λ k ,ι to λ k , n , with k > 2, and (b) fitting at least one further approximated spectrum E k (λ) to said at least one further measured extinction spectrum E k (λ), said at least one further approximated spectrum E k (λ) being a combination of a predetermined approximation function E dk (λ, a 1/S - for the background extinction, with i ranging from zero to at least one, previously determined extinction spectrums E SL (C S ,λ), with L varying from L=l to k-1, of k-1 pure components previously determined, and a predetermined extinction spectrum E sk (C Sk ,λ) of a k pure component of concentration C S k to be determined, said fitting being performed by varying the concentration Cs and at least two of the coefficients a 1/S k so that the deviation between measured spectrum and approximated spectrum is minimized, in order to determine the concentration of the second component, said at least one further selected wavelength range being so selected that the concentration C S k of said k pure component can be determined unambiguously. In a preferred embodiment the approximated spectrum E k (λ) is the sum of said predetermined approximation function E dk (λ, a 1 S k) for the background extinction, said previously determined extinction spectrums E SL (C S L,λ), with L varying from to k-1, of k-1 pure components previously determined, and said predetermined extinction spectrum Esk(C S k,λ) of said k pure component of concentration C S k. Reproducibility The reproducibility of the measured concentration C H of hemoglobin and of the measured concentration C B of bilirubin can be analytically calculated, if the minimum number N mιn = 3 of measured extinction values E(λ n ) in the range λ rh are used to determine C H from Equation (3) by means of the above described fitting step 37, and if the minimum number N mιn = 3 of measured extinction values E(λ n ) in the range λ rb are used to determine C B from Equation (5) by means of the above described fitting step 41. The reproducibility of a measured concentration C is commonly characterized by the coefficient of variation CV = σ c / E{c}, where σ x and E{x} stand for the standard deviation and the statistical expectation (mean value) of {x}, respectively. Using Equations (1), (2), and (7), it can be readily shown that the coefficient of variation CV of the concentration C-, is related to the reproducibility of the (physically) measured optical intensity σι/I 0 by the relation σ c i στ i κ ]( λ)-^-c 3+ E d (λ) CVL = —r-Xr ≡ 10 qdn , (8) lN » " E{ CJ } D I 0 C where λ is the center wavelength of the respective measurement range λ r , D = [ln(10)/4]-[2K D (λ 2 ) - K, (λi) - K- (λ 3 ) ] -[d/q dl ι] , and ln(x) is the natural logarithm of (x) . In Fig. 4, the center wavelength λ is pointed out by reference number 47. Note that the background contribution E d (λ) of the lipid and the matrix significantly reduce the reproducibility of the measured concentration C 3 . When N > N mιn statistically independent measured values E (λ n ) are used for the linear least squares algorithm, it can be shown that the coefficient of variation CV of the measured hemoglobin concentration, as well as the coefficient of variation CV of the measured bilirubin concentration, is related to Equation (8) by the relation 1 cv\ N = CV\ ( 9 ) where M = N N mιn is the number of redundant measurements. In Fig. 4, a method step 48 for computing the coefficient of variation CV of the measured hemoglobin concentration, and a method step 49 for computing the coefficient of variation CV of the measured hemoglobin concentration are represented. From Equation (9) it can be appreciated that the reproducibility of the measured concentration increases with the number N of measured extinction values considered for the curve fitting method step. The number N is given by the spectral resolution and sampling rate of the spectroscopic measurement system and the wavelength range λ r . It should be noted that an extension of the wavelength range λ r increases N for a given spectral resolution and sampling rate, but that the linear approximation E d of the sum of the lipid and the matrix contributions in Equations (3) and (5) becomes more and more inaccurate. The value of the coefficient of variation CV, calculated from Equations (8) and (9), can then be compared in a comparison step 50 with a predetermined limit value CVι ιm in order to characterize the quality of the concentration measurement. If this comparison shows that a value of CV is larger than CV llm this provides an indication 51 of a critical to weak reproducibility of the results, that is of the concentrations and the differential spectrum. Consequently, the measurement would e.g. be disregarded, repeated, or marked as being of reduced reliability. Sample quality monitoring, based on optical absorption spectroscopy as shown in Fig. 1, has been experimentally investigated using a state-of-the-art spectrometer (Cary V, VARIAN, Australia) . The collimated beam had an approximate spot size of 5 x 2 square millimeter. The optical path in the test sample was d = 10 mm. The sample dilution was 1:20 (qdii = 20) . The spectrum of the test sample was measured in the wavelength range λ = [300, 1200] nanometer with a spectral resolution of Δλ = 0.05 nanometer and a spectral sampling rate of Δλ s = 1 nanometer/pixel. The hemoglobin concentration C H has been obtained from linear least squares fitting the model in Equations (3) and (4) to N : = 28 measured values E (λ n ) in the wavelength range λ rh = [545, 575] nanometer. The bilirubin concentration C B has been obtained from linear least squares fitting the model in Equations (5) and (6) to N 2 = 63 measured values E (λ n ) in the wavelength range λ rb = [480, 545] nanometer. The differential extinction spectrum E dlff has been obtained from Equation (7) . The reproducibility of the measured hemoglobin and bilirubin concentrations has then been calculated according to Equations (8) and (9), with σ τ /I 0 = 5-10 "5 for the reproducibility of the measured optical intensities. As an example, Figure 5 shows the experimentally measured extinction spectrum E(λ) of a typical real whole blood serum 55. The best fitting extinction models for hemoglobin 57 and bilirubin 59 in Equations (3) and (5) are represented by crosses and dots, respectively. The best fitting hemoglobin and bilirubin concentrations are C H = 0.18 g/1 (CV = 1.5 %) and C B = 0.67 mg/dl (CV = 0.3 %), respectively. The differential extinction spectrum E dlff (λ n ) 60 is also shown by the dashed line. Figs. 6 and 7 show other examples of real whole blood serum samples, namely with a high hemoglobin content respectively an highly icteric sample. Furthermore, in Fig. 6, the differential spectrum shows an anomalous differential spectrum which is merely constant with additionally an increased extinction with increasing wavelength above about 650 nanometer. The continuous line 62 is the measured spectrum, the dashed line 64 and the dotted line 65 are the hemoglobin respectively the bilirubin contributions, and the dash-dotted line 66 is the differential spectrum, each time calculated from the results according to the described method. Accuracy In order to investigate the accuracy of the method, the hemoglobin and bilirubin concentrations of a series of 125 independent test samples have been determined. The samples have been synthesized using a standard blood serum (Control Serum N (human) [Hoffmann-La Roche, Switzerland] ) to which hemoglobin (Hemolysat [Hoffmann-La Roche, Switzerland] ) , bilirubin (B-4126 mixed isomers [Sigma, Switzerland] ) and lipid (Intralipid 20% [Pharmacia, Sweden] ) have been added. The added concentrations of hemoglobin, bilirubin and lipid were C H = [0, 0.17, 0.83, 3.33, 15] g/1, C B = [0, 1, 2, 10, 20] mg/dl and C L = [0, 50, 100, 400, 1800] mg/dl, respectively, leading to the set of 5-5-5 test samples. The optically measured hemoglobin 70 and bilirubin 72 concentrations versus added concentrations are represented in Fig. 8 and 9. In the case of hemoglobin (Fig. 8), a linear least squares fit 74 (c flt ,h = c 0 ,h + it-h C H ) yields an offset concentration c 0 , h = 0.12 g/1 and a slope m h = 0.95. The correlation coefficient between the best fit and the measured values is p = 0.999. In the case of bilirubin (Fig. 9), a second linear least squares fit 76 (c flt ,b = c 0 ,b + m C B ) yields an offset concentration c 0 ,b = 1.64 mg/dl and a slope m b = 0.999. The correlation coefficient between the best fit and the measured values is p = 0.995. Note that Control Serum N (human) has an approximate bilirubin concentration of C B ≡ 2 mg/dl. Further, it is stated that the amount of added hemoglobin, bilirubin and lipid also has finite accuracy. The hemoglobin and bilirubin concentrations of a series of 92 real whole blood sera have then been optically determined. The concentration values were in the range C H = [0, 5] g/1 for hemoglobin and C B = [0, 45] mg/dl for bilirubin. As reference values, the concentrations have been determined by clinical-chemical analysis (Cobas ® Integra 700 analyzer, [Hoffmann-La Roche, Switzerland] ) . Figures 10 and 11 show the optically versus clinical-chemically determined hemoglobin [bilirubin] concentrations 90 [91]. The results show that the sensitivity of the method is approximately C H ,mιn ≡ 0.5 g/1 hemoglobin and C B ,mιn ≡ 2 mg/dl bilirubin. The observed correlation coefficients between the reference 93 [94] and the optically determined 90 [91] hemoglobin [bilirubin] concentrations were p = 0.980 and p = 0.996, respectively. Note that the clinical-chemical method has also limited accuracy; namely the bilirubin concentrations (Fig. 11) show better correlation than the hemoglobin concentrations (Fig. 10) , although the accuracy of the optically measured bilirubin concentration is affected by the accuracy of the hemoglobin concentration determination (sequential determination of hemoglobin and bilirubin, see above) . In comparison, the benchmark Hitachi-Formula (US-4, 263, 512) evaluation algorithm has been used to evaluate the optical absorption spectra. The observed correlation coefficients between the reference and the Hitachi-Formula determined concentration values were p = 0.879 for hemoglobin and p = 0.992 for bilirubin. Reproducibi1ity The coefficient of variation CV of the measured hemoglobin and bilirubin concentrations has been calculated from Equation (8), where σι/I 0 = 5-10 "5 was the reproducibility of the measured optical intensities and λ = 560 nm was the center wavelength of λ rh . for hemoglobin and λ = 512 nm for bilirubin was the center wavelength of λ . Figures 12 to 15 show the optically measured hemoglobin (Fig. 12) and bilirubin (Fig. 14) concentrations of the set of 92 real blood sera and the respective CV values (Fig. 13 respectively Fig. 15). Inspection of Figs. 12 to 15 shows that the reproducibility is better for large concentration values, and that the values for hemoglobin and bilirubin are better than CV < 10 % respectively < 1 % for 89 respectively 91 of 92 analyzed sera. Low cost optical spectrometer setup Sample quality monitoring, based on optical absorption spectroscopy as shown in Fig. 1, has then been experimentally investigated with low cost optical elements. The multiple optical wavelength light source was a white- light halogen lamp (Halogen 5V, 5W, P v ≡ 2 nW/nanometer @ λ = 530 nanometer [MICROPARTS GmbH, Germany] ) . The collimated beam had an approximate diameter of D = 2 mm. The optical path in the test sample was d = 10 mm. The dilution of the sample was 1 : 20 (q dU = 20) . The transmitted light was collected by a lens (focal length f = 5 mm) and coupled into an optical fiber with core diameter 0 C = 100 μ . The light was spectroscopically analyzed by a low cost, plane-concave spectrometer PCS [CSEM-Z, Switzerland] with spectral resolution Δλ ≡ 8 nanometer. The spectrum of the test sample was measured by a linear photodiode array (512 pixels, center-to-center spacing Δx = 25 μm) in the wavelength range λ = [421, 704] nanometer. The spectral sampling rate was Δλ s = 2.8 nanometer/pixel. The reproducibility of the measured optical intensities was σ τ /I 0 = 5- 10 " ". The hemoglobin concentration C H has been obtained from linear least squares fitting the model in Equations (3) and (4) to Ni = 11 measured values E (λ n ) in the wavelength range λ rh = [545, 575] nanometer. The bilirubin concentration C B has been obtained from linear least squares fitting the model in Equations (5) and (6) to N 2 = 20 measured values E (λ n ) in the wavelength range λ rb = [480, 545] nanometer. The differential extinction spectrum E αi ff has been obtained from Equation (7) . Figures 16 and 17 show the PCS versus the state-of-the- art (Cary V) spectroscopically measured hemoglobin respectively bilirubin concentrations of the set of 92 blood sera of Figs. 10, 11 and 12 to 15. In the case of hemoglobin (Fig. 16), a linear least squares fit (Cf lt ,h = c 0 ,h + m h C H ) in the concentration range C H < 2 g/1 yields an offset concentration c 0 ,h = 0.043 g/1 and a slope m h = 0.859. The correlation coefficient between the best fitting curve and the PCS measured values is p = 0.997. In the case of bilirubin (Fig. 17), a linear least squares fit (c flt ,b = c 0 ,b + m C B ) in the concentration range C B < 15 mg/dl yields an offset concentration c 0 ,b = -0.010 mg/dl and a slope m b = 0.940. The correlation coefficient between the best fitting curve and the PCS measured values is p = 0.998. The results show that low cost spectrometers can readily be used for sample quality monitoring purposes. If the examination of the samples yields a result indicating an anomalous condition of the sample, there may be generated by the examining device, e. g., one or more of the following signals or responses: - an optical and/or acoustical warning signal to excite the operator's attention, particularly in case an abnormal sample has been detected, - a printout of results (spectra, coefficients etc.) on a printer, - a print on the analyzer's printout, so that the operator can immediately see if the results of the regular, chemical-clinical examination are true or prone to artefacts, or - an automatic repetition of the measurement, e.g. using a new test sample. The described method may be implemented in various arrangements, preferably in connection with an automated analyzer, e g. as follows: — The quality test may be done as a first photometric pass in the photometric site of an analyzer. Thereby, the performance of the analyzer is reduced because this prescan and the regular photometric pass are performed subsequently, or an additional sample is needed causing consumption of sample material; - An additional photometric site is provided for the quality test; - The pipette, or more generally, the supply system of the analyzer for the fluids to be tested, is provided with a transparent site, i. e. an optical flow-through cell (OFTC) , in connection with a photometer; where necessary, particularly when the conduit system subsequently provides differently diluted samples, there may be arranged different OFTC paths with different optical path lengths in connection with flow switches for compensating the varying dilutions; — A stand-alone photometer dedicated to the quality tests — A dip-in probe 110 having e.g. a structure of the type shown by Fig. 18. Probe 110 has a tubular body 111 and is adapted to be immersed into a liquid sample contained in a sample container. Light guides 115 and 119 are arranged within and extend along the length axis of tubular body 111. Tubular body 111 has an end part 112 and a recess 113 spaced from end part 112. Tubular body has a segment 114 of reduced cross-section. The position of this segment corresponds to the position of recess 113. Lenses 117 and 118, and a prism 116 arranged within tubular body 111 at the end 112 thereof complete the structure of dip- in probe 110. Light transmitted through light guide 115 and lens 118 impinges on and is reflected back by prism 116, traverses recess 113, where a volume of the liquid sample to be examined is present, is collected by lens 117 and is transmitted to a photometer via light guide 119. - The sample tube itself may be used as the photometric cuvette, provided the differing lengths of the optical observation paths can be compensated for, i. e. the path lengths are determined and can be input into the quality test system, and/or the sample tubes are of sufficiently equal size so that the optical paths do only differ within small limits, maybe in an even negligible variation range. From the above description, variations of the invention are conceivable to the one skilled in the art without leaving the scope of the invention as defined in the claims. For instance, it is conceivable: - to extend the method to the determination of a third and further components by continuing the sequential determination method using two, three, four etc. previously determined components for ascertaining the concentrations of a third, fourth etc. component; - to have the differential spectrum analyzed automatically by determining its curvature (i. e. the second derivative) and/or slope (i. e. the first derivative), which should increase respectively be negative for increasing wavelength in the exemplary quality test set forth above; - to choose deviating wavelength ranges for the photometric measurements, particularly if the quality test is used for determining other components of the samples provided that the spectra to be combined in order to approximate the measured spectrum show peculiarities in the given wavelength range so that the approximation parameters, before all the concentration of the sought component, are unambiguously derivable; - to determine the differential spectrum in a subrange of the wavelength range used for the determination of the single components, or possibly even a range extending beyond this range. List of reference numbers 1 beam of light 2 light source (multiple wavelenght) 3 lens 4 lens 5 input of wavelength analyzer 10 extinction coefficient K H of hemoglobin 11 extinction coefficient K H of bilirubin 12 extinction coefficient K L of lipid 13 extinction coefficient E g of a standard blood serum 30 extinction spectra of the reference solution of Intralipid 31 extinction spectra of several samples of real whole blood sera 32 method step: determination of hemoglobin concentration 33 method step: determination of bilirubin concentration 34 method step: determination of E dlff and further evaluation 35 measured extinction spectrum E(λ) 35 in the approximate wavelength range λ rh ≡ [545, 575] nm 36 determination of E d ι at λ = 560 nm 37 method step: curve fitting 38 set of values: C H and a 0 h, a ih 40 determination of E dX at λ = 512 nm 41 method step: curve fitting 42 set of values: C B and a 0 b, a ib 45 determination of E dlff 46 method step: investigation for spectral anomalies 46 47 center wavelength 48 computation of the coefficient of variation CV of the measured hemoglobin concentration 49 computation of the coefficient of variation CV of the measured bilirubin concentration 50 comparison step 51 indication of a critical to weak reproducibility of the results 55 measured extinction spectrum E (λ) of a typical real whole blood serum 57 best fitting extinction model for hemoglobin 59 best fitting extinction models for bilirubin 60 differential extinction spectrum E dlff (λ n ) 62 measured spectrum 64 hemoglobin contribution 65 bilirubin contribution 66 differential spectrum 70 optically measured hemoglobin concentration 72 optically measured bilirubin concentration 74 linear least squares fit 76 linear least squares fit 90 optically determined hemoglobin concentrations 91 optically determined bilirubin concentrations 93 clinical-chemically determined hemoglobin concentrations 94 clinical-chemically determined bilirubin concentrations 96 state-of-the-art (Cary V) spectroscopically measured hemoglobin concentrations 97 linear least squares fit 99 state-of-the-art (Cary V) spectroscopically measured bilirubin concentrations 100 linear least squares fit 110 dip-in probe 111 tubular body 112 end part of tubular body 111 113 recess 114 segment having a reduced cross-section 115 light guide 116 prism 117 lens 118 lens 119 light guide

Claims: Claims 1. Method for measuring the concentration of at least one component of a liquid biological sample before analysis of said sample by an in vitro diagnostic method, said component being apt to interfere with the measurement of a target analyte by means of said diagnostic method, said measuring method comprising (a) measuring a first extinction spectrum E x (λ) of said liquid sample in a first selected wavelength range (b) fitting an approximated spectrum E^λ) to said first measured extinction spectrum Ei (λ) , said approximated spectrum E^λ) being a combination of a predetermined approximation function E d ι (λ, a lrS ι) for the background extinction, with i ranging from zero to at least one, and a predetermined extinction spectrum E Ξι (C Sι ,λ) of a first pure component of concentration C S ι of the components to be determined, said fitting being performed by varying said concentration Csi of said first interfering component and at least two of said coefficients so that the deviation between said first measured extinction spectrum Ei (λ) and said approximated spectrum E^tλ) is minimized in order to determine the concentration of said first interfering component, and said first selected wavelength range being so selected that the concentration C S ι of said first interfering component can be determined unambiguously. 2. A method according to claim 1, wherein said approximated spectrum E ] _(λ) is the sum of said predetermined approximation function E d ι(λ,a 1#sι ) for the background extinction, and said predetermined extinction spectrum E S ι(C S ι,λ) of said pure first component of concentration C S ι. 3. A method according to claim 1 or 2, characterized in that it further comprises (a) measuring at least one further extinction spectrum E 2 (λ) of said liquid sample in at least one further selected wavelength range λ= λ i to λ k , n , with k > 2, and (b) fitting at least one further approximated spectrum E2(λ) to said at least one further measured extinction spectrum E 2 (λ) , said at least one further approximated spectrum Eι(λ) being a combination of a predetermined approximation function E dk (λ, a 1(S ) for the background extinction, with i ranging from zero to at least one, previously determined extinction spectra E SL (C SL ,λ), with L varying from L=l to k-1, of k-1 pure components previously determined, and a predetermined extinction spectrum E Sk (C Sk ,λ) of a k pure component of concentration C S k to be determined, said fitting being performed by varying the concentration C Sk and at least two of the coefficients a 1/Sk so that the deviation between measured spectrum and approximated spectrum is minimized, in order to determine the concentration of the second component, said at least one further selected wavelength range being so selected that the concentration C S k of said k pure component can be determined unambiguously. 4. A method according to claim 3, wherein said approximated spectrum E k (λ) is the sum of said predetermined approximation function E dk (λ, a x , sk ) for the background extinction, said previously determined extinction spectra E S (Cs/λ), with L varying from L=l to k-1, of k-1 pure components previously determined, and said predetermined extinction spectrum E Sk (C Sk ,λ) of said k pure component of concentration C Sk . 5. A method according to claims 1 to 4, characterized in that at least one , and preferably all, of the functions E d (λ,ai,s ) with k > 1, are of the form n f] (λ, a i(k )= a ifk λ 1 i=0 with n > 1 and preferably n=l . 6. A method according to one of claims 1 to 5, characterized in that said fitting of said approximated spectra E k (λ) , with k > 1, to said the measured values of extinction spectra E(λ_), with i=l to N, N being the number of measured values, is done by a least squares fitting method. 7. A method according to one of claims 1 to 6, characterized in that the sample is marked at least anomalous if the determined concentrations C S k with k > 1 are outside a predetermined range. 8. A method according to one of claims 1 to 7, characterized in that a differential spectrum j Ediff( λ ) = E(λ) - ∑ EjtCj λ) , J = l with J being the number of components, and λ being in a range covering at least 30 %, preferably at least 50 %, and most preferably about 100 % or more of the whole wavelength range defined by the broadest combination of λι,ι and λι, n , λ 2 ,ι and λ 2 , n ,-, λ J# i and λj, n is computed, and the differential spectrum is subjected to an analysis in view of anomalies. 9. A method according to claim 8, characterized in that the curvature and/or the slope of the differential spectrum in at least one predetermined wavelength range is/are determined, the result compared with the expected values, and in that the differential spectrum is estimated to be normal if the values compared have identical sign, optionally with the magnitude resting in a predetermined range given by an upper and a lower limiting curve. 10. A method according to one of claims 1 to 9, characterized in that the sample is blood, preferably human blood, or a fluid derived therefrom, the first wavelength range is chosen in the range of 500 to 600 nanometer, preferably from 545 to 575 nanometer, even more preferably being essentially identical with one of these ranges, the first reference spectrum E]_ (λ) being that of hemoglobin, so that the concentration C of hemoglobin is determinable, and the second wavelength range is chosen in the range of 400 to 600 nanometer, preferably from 480 to 545 nanometer, even more preferably being essentially identical with these ranges, the second reference spectrum E2 (λ) being that of bilirubin, so that the concentration C 0 of bilirubin is determinable . 11. A method according to claim 10, characterized in that the lipid concentration and the overall constitution of the sample are estimated to be normal if the differential spectrum has a negative slope and/or a positive curvature. 12. A method according to one of claims 10 to 11, characterized in that the sample is estimated to be of critical condition if the concentration of bilirubin and/or hemoglobin exceed a predetermined value, and/or if the differential spectrum is anomalous. 13. A method according to one of claims 1 to 11, characterized in that the spectra are provided as electrical signals and furnished to an evaluation device comprising a processor which performs the method steps on the spectra under the control of a program, and that the results are stored in a storage means, preferably a storage means for digital data, and/or presented to an operator, preferably by printing, displaying and/or producing audible sounds. 14. An installation for implementing the method of one of claims 1 to 13 for use with an analyzer, preferably a clinical-chemical analyzer, characterized in that in the supply path of sample fluid of the analyzer, at least one photometric measurement site is provided so that extinction spectra can be taken of the fluid in the supply path. 15. A photometric probe for implementing the method of one of claims 1 to 13, characterized in that its end comprises a photometric measurement site confined by two facing walls, one of which being equipped with a light source, and the second well being equipped with a light capturing means, the measurement site, the light source and the light capturing means being so arranged that light emanating from the light source passes the measurement site and, at least to a significant part, is captured by the light capturing means. 16. A photometric probe according to claim 15, characterized in that it comprises a light guide passing the measurement site, and that a light deviating means, preferably a prism, is arranged such that light exiting the light guide is RECTIFIED SHEET (RULE 91) ISA/EP deviated, preferably by an angle of substantially 180°, towards the light exiting side of the first wall of the measurement site. 17. An analyzer, preferably a chemical-clinical analyzer, with an installation for photometric measurements, characterized in that the installation comprises a program memory and a device for executing the program, wherein the execution of the program implements the method of one of claims 1 to 12. RECTIFIED SHEET (RULE 91) ISA/EP

Inventors: ELSENHANS OLIVIER, SAROFIM EMAD, SCHNELL URBAN GEORG, ELSENHANS, OLIVIER, SAROFIM, EMAD, SCHNELL, URBAN, GEORG
Applicants: ELSENHANS OLIVIER, HOFFMANN LA ROCHE, SAROFIM EMAD, SCHNELL URBAN GEORG, ELSENHANS, OLIVIER, F. HOFFMANN-LA ROCHE AG, SAROFIM, EMAD, SCHNELL, URBAN, GEORG
Application year: 2000
Main classification: G01N21
IPC codes: G01N21, G01N33