Method for determining lipids and other interfering substances in body fluid samples

Abstract

The present invention relates to a method and an automatic analyzer for determining more accurately the concentration of lipids and other interfering substances in body fluids, particularly of interfering substances such as bilirubin and hemoglobin in blood serum and blood plasma samples.

Claims

1. A method for determining the concentration of lipids in a body fluid sample, comprising the steps: a) transirradiating the body fluid sample with light at a multiplicity of wavelengths; b) capturing a first measurement value (A1) at a first wavelength at which the absorbance not caused by lipids is negligible; c) capturing a second measurement value (A2) at a second wavelength at which bilirubin has an absorbance maximum; d) capturing a third measurement value (A3) at a third wavelength at which hemoglobin has an absorbance maximum; e) capturing a fourth measurement value (A4) at a fourth wavelength at which the absorbance not caused by bilirubin and hemoglobin and lipids is negligible; f) calculating a power-function approximation curve (L.sub.0) of the form
E()=p.sub.0.Math..sup.q.sup.0 for the absorbance of the lipids on the basis of the first measurement value (A1) by determining the factor p.sub.0 at predetermined exponent q.sub.0; g) determining an approximation value of the bilirubin concentration (c.sub.B) on the basis of a first theoretical absorbance value (E.sub.B) for bilirubin, corresponding to the difference between the second measurement value (A2) and the value of the approximation curve (L.sub.0) at the second wavelength; h) determining an approximation value of the hemoglobin concentration (c.sub.H) on the basis of a second theoretical absorbance value (E.sub.H) for hemoglobin, corresponding to the difference between the third measurement value (A3) and the value of the approximation curve (L.sub.0) at the third wavelength; i) determining a third theoretical absorbance value (E.sub.HBL) for the fourth wavelength on the basis of the sum of the theoretical absorbance values for hemoglobin (E.sub.H) and bilirubin (E.sub.B) and the value of the approximation curve (L.sub.0) at the fourth wavelength; j) ascertaining a deviation of the third theoretical absorbance value (E.sub.HBL) from the fourth measurement value (A4), wherein: I.if the deviation ascertained in step j) does not exceed a predetermined threshold valuethe concentration (c.sub.L) of lipids is determined by generating the difference between the value of the approximation curve (L.sub.0) at the fourth wavelength and the value of the approximation curve (L.sub.0) at the first wavelength and dividing said difference by the extinction coefficient specific for lipids, or II.if the deviation ascertained in step j) exceeds a predetermined threshold valuea corrected approximation curve (L.sub.k) for the absorbance of the lipids is calculated and steps g) to j) are repeated with the values of the corrected approximation curve (L.sub.k) until the deviation reaches or falls short of the predetermined threshold value, and the concentration (c.sub.L) of lipids is determined by i.if the first measurement value (A1) does not exceed a predetermined absorbance threshold value for the absorbance at the first wavelengthgenerating the difference between the value of the corrected approximation curve (L.sub.k) at the fourth wavelength and the value of the approximation curve (L.sub.k) at the first wavelength and dividing said difference by the extinction coefficient specific for lipids or ii.if the first measurement value (A1) exceeds a predetermined absorbance threshold value for the absorbance at the first wavelength and if the exponent q.sub.k of the corrected approximation curve is greater than 1correcting the value of the corrected approximation curve (L.sub.k) at the first wavelength by means of an equalizing function which relates the first measurement value (A1) to the value of the corrected approximation curve (L.sub.k) at the first wavelength, and then generating the difference between the value of the corrected approximation curve (L.sub.k) at the fourth wavelength and the value at the first wavelength, as corrected by application of the equalizing function, and dividing said difference by the extinction coefficient specific for lipids.

2. The method as claimed in claim 1, wherein the first wavelength is within the range between 600 nm and 660 nm, and the second wavelength is within the range between 440 nm and 480 nm, and the third wavelength is within the range between 400 nm and 440 nm, and the fourth wavelength is within the range between 350 nm and 370 nm.

3. The method as claimed in claim 2, wherein the first wavelength is 645 nm or the second wavelength is 470 nm or the third wavelength is 415 nm or the fourth wavelength is 365 nm.

4. The method as claimed in claim 1, wherein the concentration of hemoglobin (c.sub.H) or the concentration of bilirubin (c.sub.B) is additionally determined.

5. The method as claimed in claim 4, wherein, if the deviation ascertained in step j) does not exceed a predetermined threshold value, the concentrations (c.sub.H, c.sub.B) of hemoglobin and bilirubin are determined by outputting the approximation values of the hemoglobin concentration and the bilirubin concentration, as determined in steps g) and h), as concentrations (c.sub.H, c.sub.B) of hemoglobin and bilirubin.

6. The method as claimed in claim 3, wherein, if the deviation ascertained in step j) exceeds a predetermined threshold value, the concentrations (c.sub.H, c.sub.B) of hemoglobin and bilirubin are determined by outputting the approximation values of the hemoglobin concentration and the bilirubin concentration, as determined in steps g) and h) that are repeated with the values of the corrected approximation curve (L.sub.k), as concentrations (c.sub.H, c.sub.B) of hemoglobin and bilirubin when the deviation reaches or falls short of the predetermined threshold value.

7. The method as claimed in claim 1, wherein the predetermined threshold value is 10 mAU.

8. The method as claimed in claim 1, wherein the predetermined absorbance threshold value for the absorbance at the first wavelength is 950 mAU.

9. The method as claimed in claim 1, wherein a transirradiation of the body fluid sample with light at a multiplicity of wavelengths is achieved using laser or light-emitting diodes or using a light source having various optical filters and wherein the capture of the multiplicity of measurement values (A1; A2; A3; A4) is achieved using a photodetector.

10. The method as claimed in claim 1, wherein the body fluid sample is serum or plasma.

11. An automatic analyzer comprising a measuring device designed to carry out method steps a) to e) as claimed in claim 1, characterized in that the analyzer further comprises a calculation device designed to carry out the remaining method steps for determining the concentration (c.sub.L) of lipids as claimed in claim 1.

12. The automatic analyzer as claimed in claim 11, wherein the measuring device comprises at least one light source and multiple optical filters.

13. The automatic analyzer as claimed in claim 11, wherein the measuring device comprises multiple light sources, preferably multiple light-emitting or laser diodes.

14. The automatic analyzer as claimed in claim 13, wherein the measuring device comprises at least four light sources, wherein the first light source emits light of a wavelength within the range between 600 nm and 660 nm, and the second light source emits light of a wavelength within the range between 440 nm and 480 nm, and the third light source emits light of a wavelength within the range between 400 nm and 440 nm, and the fourth light source emits light of a wavelength within the range between 350 nm and 370 nm.

15. The automatic analyzer as claimed in claim 14, wherein the first light source emits light of a wavelength of 645 nm, and the second light source emits light of a wavelength of 470 nm, and the third light source emits light of a wavelength of 415 nm, and the fourth light source emits light of a wavelength of 365 nm.

16. The automatic analyzer as claimed in claim 11, wherein the measuring device comprises at least one photodetector.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a schematic representation of a graph containing the absorbance curve of a plasma sample. The absorbance curve (solid line) with the measurement values A1=A.sub.645, A2=A.sub.470, A3=A.sub.415 and A4=A.sub.365 reflects an exemplary schematic profile for the wavelength-dependent absorbance of human plasma with concentrations of hemoglobin, bilirubin and lipids, the respective absorbances of which overlap additively. The approximation curve L.sub.0 (dotted line) for the absorbance of the lipids is calculated on the basis of the first measurement value A1=A.sub.645 and the power function E()=p.sub.0.Math..sup.%. The approximation curve L.sub.k (dashed line) is calculated and ultimately used for calculating the lipid concentration and the hemoglobin and bilirubin concentrations. The absorbance values of the measured plasma sample are comparatively low, and as a result, the approximation curve L.sub.0 approximates the absorbance spectrum from below by iteration until it reaches the final approximation curve L.sub.k.

(2) FIG. 2 shows, like FIG. 1, a schematic representation of a graph containing the absorbance curve of a plasma sample. In comparison with the sample from FIG. 1, the measured absorbance values in this case are comparatively high, and as a result, the approximation curve L.sub.0 approximates the absorbance spectrum from above by iteration until it reaches the final approximation curve L.sub.k.

(3) FIG. 3 shows a graph concerning the schematic representation of the comparison of the true lipid content ascertained by chemical analysis (X-axis) and the lipid content ascertained using the method according to the invention (Y-axis) for multiple human plasma samples. It is clear that the lipid content determined according to the invention does not deviate in any case by more than +/20% from the true lipid content.

(4) FIG. 4 shows a graph concerning the schematic representation of the comparison of the lipid determination (Y-axis) by means of chemical analysis (diamonds), by means of the method according to the prior art from WO 2013/010970 A1 (triangles) and by means of the method according to the invention (squares) in plasma samples having various bilirubin concentrations (X-axis). It is clear that the lipid content determined according to the invention substantially agrees with the lipid content determined chemically.

(5) FIG. 5 shows a graph concerning the schematic representation of the comparison of the lipid determination (Y-axis) by means of chemical analysis (diamonds), by means of the method according to the invention without equalizing function (squares) and by means of the method according to the invention with equalizing function (triangles) in 11 plasma samples having high lipid concentrations (X-axis). It is clear that the determination according to the invention with equalizing function shows a good agreement with the chemical analysis.

DETAILED DESCRIPTION

Example

(6) a) Wavelengths

(7) The method according to the invention was carried out in an automatic analyzer comprising a photometric arrangement having four laser diodes. Human plasma samples were transirradiated with light of the following wavelengths:

(8) 645 nm first wavelength at which the absorbance not caused by lipids is negligible;

(9) 470 nm second wavelength at which bilirubin has an absorbance maximum;

(10) 415 nm third wavelength at which hemoglobin has an absorbance maximum;

(11) 365 nm fourth wavelength at which the absorbance not caused by bilirubin and hemoglobin and lipids is negligible.

(12) The above-mentioned four measurement values (A2=A.sub.645, A2=A.sub.470, A3=A.sub.415 and A4=A.sub.365) were recorded.

(13) b) Lipid-Specific Extinction Coefficient

(14) The lipid-specific extinction coefficient .sub.Lipid was ascertained by measuring the absorbance spectrum using the above-mentioned wavelengths for 70 plasma samples of known lipid content ascertained by chemical analysis (L.sub.1, L.sub.2, . . . L.sub.n), calculating the power-function approximation curve (L.sub.0) of the form
E()=p.sub.0.Math..sup.q.sup.0
for the absorbance of the lipids using a predetermined exponent q.sub.0=2.46, and generating the value of the approximation curve (L.sub.0) at the fourth wavelength (E.sub.365) and the value of the approximation curve (L.sub.0) at the first wavelength (E.sub.645). The various absorbance values were calculated on the basis of the corrected approximation curve L.sub.k (see point c) below).

(15) The difference between the values E.sub.365 and E.sub.645 divided by the lipid concentration yields the specific extinction coefficient for lipid. The mean and the median were generated across all measurements:

(16) ${\mathrm{.Math.}}_{\mathrm{Lipid}}=\frac{1}{n}\underset{i=1}{\overset{n}{.Math.}}\mathrm{.Math.}=\frac{\left[\frac{E_{1_{365}}-E_{1_{645}}}{L_1}\right]+\left[\frac{E_{2_{365}}-E_{2_{645}}}{L_2}\right]+\left[\frac{E_{n_{365}}-E_{n_{645}}}{L_n}\right]}{n}$

(17) The result is the lipid-specific extinction coefficient .sub.Lipid=0.0010 dL/mg.

(18) c) Establishing the Power-Function Approximation Curve for the Absorbance of the Lipids and Calculating the Concentrations of Lipids and of Hemoglobin and Bilirubin

(19) The power-function approximation curve (L.sub.0) of the form
E()=p.sub.0.Math..sup.q.sup.0
for the absorbance of the lipids is calculated on the basis of the first measurement value (A1=A.sub.645) by determining the factor p.sub.0 at predetermined exponent q.sub.0.

(20) It is self-evident that solely the measurement value A.sub.645 cannot yet be used for a determination of the two variables p and q. Therefore, the exponent q.sub.0 can be generated on the basis of an estimation based on reference values. The exponent q.sub.0 can thus be predetermined on the basis of empirical values. In the case of the determinations described here, an exponent q.sub.0=2.46 was predetermined. Since, at the first wavelength of 645 nm, the absorbance due to substances other than lipids can be ignored, it is possible to determine, at given exponent q.sub.0, the coefficient p.sub.0 via the measurement value A.sub.645 at the first wavelength. The thus ascertained approximation curve having the parameters p.sub.0 and q.sub.0 can reflect a first approximation for the absorbance profile of the absorbance of lipids in the sample. Concerning this, it is possible, in the case of all wavelengths in which further measurement values have been captured, to calculate the particular share of the absorbance of the lipids.

(21) The result of this is a first approximation curve L.sub.0 which already provides a good approximation of the actual lipid absorbance. However, the approximation curve L.sub.0, particularly within a blue or ultraviolet spectral range, may run flatter than the actual absorbance profile for lipids.

(22) First approximation values for the concentrations of bilirubin (c.sub.B) and hemoglobin (c.sub.H) are then determined on the basis of the measurement values A2=A.sub.470 and A3=A.sub.415 at the wavelengths 470 nm and 415 nm:

(23) $c_B=\frac{A_{470}-c_H.Math.{\mathrm{.Math.}}_{H470}-E_{L470}}{{\mathrm{.Math.}}_{B470}}$$c_H=\frac{A_{415}-c_B.Math.{\mathrm{.Math.}}_{B415}-E_{L415}}{{\mathrm{.Math.}}_{H415}}$
where .sub.H470, .sub.H415, .sub.B470 and .sub.B415 are the respective extinction coefficients of bilirubin (B) and hemoglobin (H) at the wavelengths of the measurement values A.sub.470 and A.sub.415. In this connection, the extinction coefficients can be determined in advance by reference measurements, or be retrieved for the calculations from a storage device in which reference values have been stored.

(24) The two concentrations c.sub.B and c.sub.H can be ascertained by solving the linear system of the two aforementioned equations, yielding the formula

(25) $c_H=\frac{A_{470}-E_{L470}-\frac{\left(A_{415}-E_{L415}\right).Math.{\mathrm{.Math.}}_{B470}}{{\mathrm{.Math.}}_{B415}}}{{\mathrm{.Math.}}_{H470}-\frac{{\mathrm{.Math.}}_{H415}.Math.{\mathrm{.Math.}}_{B470}}{{\mathrm{.Math.}}_{B415}}}$
for the concentration of hemoglobin (H). Here, it is possible to determine the absorbance values for the lipids, E.sub.L470 and E.sub.L415, using the approximation curve L.sub.0. The result is therefore a first approximation value for the concentration c.sub.H of hemoglobin. Said first approximation value for the concentration c.sub.H can then be used for determining the first approximation value for the concentration c.sub.B of bilirubin. This already yields first, good approximation values c.sub.H, c.sub.B and c.sub.L for the concentrations of hemoglobin, bilirubin and lipids, which values were ascertained on the basis of the power function and the above-described linear system with the first approximation values for the parameters p.sub.0 and q.sub.0.

(26) However, the approximation values can now be improved further iteratively, as will be described below. To this end, a theoretical absorbance value (E.sub.HBL) for the fourth wavelength is determined on the basis of the sum of the theoretical absorbance values for hemoglobin (E.sub.H) and bilirubin (E.sub.B) and of the value of the approximation curve (L.sub.0) at the fourth wavelength (365 nm), i.e., within a range in which a relatively high deviation of the actual lipid absorbance from the approximation curve is to be expected:
E.sub.HBL=c.sub.H.Math..sub.H365+c.sub.B.Math..sub.B365+E.sub.L365

(27) The concentrations c.sub.H and c.sub.B were determined above; the value E.sub.L365 (E.sub.L4) is again yielded by the power function with the parameters p.sub.0 and q.sub.0.

(28) Then, a comparison between the value E.sub.HBL and the actual measurement value A4=A.sub.365 at this wavelength is carried out in order for a deviation (DeltaE)
E=A.sub.365E.sub.HBL
to be obtained. If the deviation E (DeltaE) is greater than a predetermined threshold value, for example 10 mAU, it can be determined that the ascertained approximation curve L.sub.0 for the concentrations of the lipids has not been ascertained sufficiently accurately enough. In this case, a correction of the approximation curve L.sub.0 can be carried out in a further step. To this end, the calculated absorbance value E.sub.L365 (E.sub.L4), which describes the lipid share of absorbance at the wavelength 365 nm, can be corrected by a percentage of the deviation E. For example, half the value of the deviation E can be added to the absorbance value E.sub.L365 (E.sub.L4). On the basis of the corrected absorbance value E.sub.L365 (E.sub.L4), it is then possible to determine a corrected approximation curve L.sub.k with the parameters p.sub.k and q.sub.k:

(29) $q_k=\frac{\ln E_{L645}-\ln \left(E_{L365}+E/2\right)}{\ln {}_{645}-\ln {}_{365}}$$p_k=\frac{E_{L365}}{{}_{365}^{q_k}}$

(30) The equations arise here by inserting the values of E.sub.L365 and the corrected value E.sub.L365+E/2 into the power function. This means that the approximation curve L.sub.0 can be corrected such that the measurement value A1=A.sub.645 at the wavelength of 645 nm continues to lie on the corrected approximation curve L.sub.k, i.e., the measurement value A.sub.645 is used as anchor point for the approximation curve.

(31) In the next step, the various shares of absorbance of the lipids are then calculated on the basis of the corrected approximation curve L.sub.k. The method is iterated until it is determined that the deviation falls short of the predetermined threshold value. The corrected approximation values for the concentrations of hemoglobin, bilirubin and lipids in the body fluid sample are then outputted.

(32) Using the described method, the lipid content in all the samples was determined with a deviation of not more than +/20% from the true lipid content (see FIG. 3).

(33) d) Determining the Lipid Content in Turbid Samples

(34) Plasma samples which are frozen and thawed frequently exhibit a turbidity which can result in an elevated absorbance.

(35) It has been found that, in contrast to the method known from WO 2013/010970 A1, it is possible to carry out a lipid determination independently of the turbidity using the method according to the invention.

(36) The lipid content of a plasma sample (No. 2004657355) of known lipid concentration (667.7 mg/dL) was determined using the method according to the prior art from WO 2013/010970 A1 and the method according to the invention. The sample was then frozen, thawed after six months, and the lipid content was determined again. The total rise in absorbance of the thawed sample at the wavelengths used was 115%, i.e., the sample was more turbid after thawing than before freezing. The results are shown in Table 1. It can be seen from the table that, with the conventional method, the lipid content measured after thawing is more than twice that before freezing, but still deviates by more than 20% from the true lipid content, whereas with the method according to the invention, only a 5% deviation is measured after thawing with respect to before freezing. Moreover, the deviation from the true value of the lipid concentration is merely 4.7% or 0.4%, i.e., the method according to the invention is in principle also more accurate.

(37) TABLE-US-00001 TABLE 1 Before freezing After thawing Lipid determination according to prior art Concentration 227.8 mg/dL 489.9 mg/dL Relative deviation of 66% 27% the concentration from the true value (667.7 mg/dL) Relative deviation of +115% the concentration after thawing Lipid determination according to invention Concentration 636.6 mg/dL 670.6 mg/dL Relative deviation of 4.7% .sup.0.4% the concentration from the true value (667.7 mg/dL) Relative deviation of +5% the concentration after thawing

(38) e) Determining the Lipid Content in the Presence of High Bilirubin Concentrations

(39) Various amounts of bilirubin were added to plasma samples so that 9 visually distinguishable concentration levels were obtained, and the lipid content was determined by chemical means, using the method according to the prior art from WO 2013/010970 A1 and using the method according to the invention. The results are shown in FIG. 4. It is clear from this that, in the presence of various bilirubin concentrations, the lipid determination according to the invention works virtually just as well as the determination by chemical means; the greatest deviation is merely 7% (concentration level 9).

(40) f) Determining the Lipid Content in Highly Turbid Samples

(41) Samples of very high turbidity, for example because of a very high lipid content (>500 mg/dL), have a high total absorbance. Therefore, in the case of some samples, a sufficiently accurate lipid determination is not possible using the method according to the invention, as described as before. The reason therefor is that, with the increase in lipid concentration, the absorbance spectrum of the sample firstly rises evenly, but then tips asymmetrically because the increase in absorbance is greater at high wavelengths than at lower wavelengths. As a result of this, the difference in absorbance between the first wavelength (645 nm) and the fourth wavelength (365 nm), which are used in the method according to the invention, is no longer large enough in order to be able to use the method to determine the lipid content with sufficient accuracy. Underdeterminations of up to 70% were observed.

(42) However, this effect is correctable by means of an equalizing function. The equalizing function corrects the value of the corrected approximation curve (L.sub.k) at the first wavelength (E.sub.645). The equalizing function is ascertainable using a number of samples of known lipid concentration. By back-calculation of the concentration into the expected absorbance, it is possible to determine an equalizing function which allows calculation of the true lipid content.

(43) Since turbidity is heavily dependent on absorbance at the wavelength 645 nm, the share of turbidity at 645 nm can be taken into account.

(44) The calculation is formed as follows:
DeltaE=E.sub.L365(0.7798.Math.E.sub.L645+1,4626).Math.E.sub.L645|

(45) The result of the application of this function to the first measurement value (A1=A.sub.645) of the samples and the ensuing division of the difference (Delta E) between the value of the corrected approximation curve (L.sub.k) at the fourth wavelength (365 nm) and the value at the first wavelength (645 nm), as corrected by application of the equalizing function, by the extinction coefficient specific for lipids is that, even in the highly turbid samples, the lipid concentration can be determined with sufficient accuracy, with a deviation from the true value of less than +/20%. FIG. 5 shows a comparison of the lipid determinations for 11 plasma samples having high lipid concentrations.