MXPA98009703A

MXPA98009703A - Method for the analysis of medical samples containing hemoglob

Info

Publication number: MXPA98009703A
Application number: MXPA/A/1998/009703A
Authority: MX
Inventors: Schellong Lieselotte; Weisheit Ralph
Original assignee: Boehringer Mannheim Gmbh 68305 Mannheim De
Priority date: 1996-05-31
Filing date: 1998-11-19
Publication date: 1999-09-20

Abstract

The present invention relates to a method for the determination of an analyte in a sample containing free hemoglobin in which the determination is carried out by an optical measurement and the value measured for the concentration of the analyte is corrected mathematically. This method is particularly suitable for determining the parameters of total protein, iron and albumin in a medical sample, for example, in a serum or plasma sample. The correction of the measured value for the concentration of the analyte is made by steps (a) measuring the assumed value of the sample being analyzed, (b) measuring the assumed value of a reference sample free of hemoglobin, (c) measuring the incorrect value for the concentration of the analyte and (d) correct the value obtained in step (c) by correlation with the values obtained in step (a) and (b) to obtain the corrected value for the concentration of the analyte.

Description

METHOD FOR THE ANALYSIS OF MEDICAL SAMPLES CONTAINING HEMOGLOBIN The invention relates to a method for the determination of an analyte in a sample containing free hemoglobin, in which the determination is carried out by an optical measurement and the value measured for the concentration of the analyte is mathematically corrected. In particular, this method is suitable for the determination of the parameters of total protein, iron and albumin in a medical sample, for example, in a serum or plasma sample.

It is generally known that hemolysis interferes in some cases to a considerable degree with the determination of numerous analytes. However to obtain measurement values that are not altered, several methods have been published in the past for the reduction of interference by hemolysis As mentioned in EP-0 268 025 Bl, a graphic relationship is established for some analytes between the degree of hemolysis and the resulting measurement error. The correction factors could be derived from this, which are used to mathematically correct the result Ref. 028723 analytical obtained on the basis of a separate determination of the degree of hemolysis.

Jay and Provasek also describe that unaltered values can be obtained in haemolytic samples by determining the degree of hemolysis and using a correction factor (Clin Chem. 38/6, 1026 (1992) and Clin. Chem. 39/9, 1804 -1810 (1993)). In this case the degree of hemolysis is determined by a separate measurement of the Hb content in the sample.

In the patent document US 4,263,512 it is recommended that the degree of turbidity (X), hemolysis (Y) and jaundice (Z) are determined in addition to the analyte and that the measured value of the analyte (S) is corrected with the help of the formula S '= S - aX - ß - Y -? - Z. In this case S1 is the corrected value of the analyte and a, ß and y are correction factors which are obtained by measuring the influence of turbidity, hemolysis and jaundice by means of reference liquids. X, Y and Z are determined by multichannel measurement and a subsequent complicated calculation from the absorbance differences obtained taking into account the respective proportion of the other interfering substances.

A method of correction of interference by haemolysis without a separate determination of the degree of hemolysis is shown by DE 44 27 492 A1. Here is a mathematical relationship between the content of interference substance released by the hemolysis of erythrocytes and a prereaction which occurs before the main reaction. The analytical result obtained in the main reaction (total ratio) can be corrected with the help of the degree of hemolysis determined in this way during the prereaction using the ratio found between the degree of hemolysis and the interference contribution according to the formula ratio subsample / sample = total ratio - prerepair ratio -substance ratio / erythrocyte , in which the substance means the component that is determined in the sample.

Frequently an interference by hemoglobin can also be eliminated by measuring the assumed value of the sample. This, however, does not apply to the determination of total protein by means of the Biuret method (Morgan et al., Microchem J. 44, 282-287 (1991)) and also does not apply to the determination of albumin by of bromocresol-green and bromocresol-purple method. It is also known that interference by hemoglobin always occurs in the determination of iron if the Hb of the sample has not been previously eliminated by dialysis (Sonntag, J. Clin. Chem. Clin. Biochem. 24/2, 127-139 (1986)). Also in the case of iron, it is not possible to eliminate the interference of the Hb by the only measurement of the assumed value of the sample.

However, all the methods for eliminating the interference described above have the disadvantage that it comprises a considerable amount of work (preparation of the sample by dialysis or the separate determination of the degree of hemolysis, for example determining the Hb content) and / or algorithms. of complicated mathematical correction.

In addition all the described methods refer to the elimination of erroneous measurements caused by hemolysis. The development of blood substitutes based on hemoglobin has even made the problem of elimination of interference by natural or synthetic hemoglobin or hemoglobin-like compounds more critical than the previous one. On the other hand then also such interference occurs in a non-haemolytic sample material and on the other hand also to a much greater extent than in the case of natural haemolysis since in the treatment of the blood substitute, the hemoglobin content in the Serum or blood plasma can be greater than 1000 mg / dl.

The aim of the present invention is to provide a method for the elimination of interferences which are caused by natural hemoglobin or blood substitutes based on synthetic Hb or Hb-like compounds and which can not be eliminated by simple value measurement course of the sample. In addition, this method must be associated with a significantly reduced workload compared to conventional methods and guarantees an elimination of interference up to at least 1000 mg / dl of Hb.

The object of the invention is achieved since surprisingly there is a relationship between the level of the assumed value of the sample and the degree of alteration of the measured results caused by the free hemoglobin. As a result it is possible with the help of a simple mathematical correction formula to accurately determine the correct value for the concentration of the analyte even when the Hb content is high. In comparison with the statement of the technique, the method according to the invention is advantageously characterized since it is not necessary to determine the Hb content separately or a determination of the degree of a prereaction to correct the measured value of a medical sample. which contains Hb.

Here a subject of the present invention is a method for the determination of an analyte in a sample containing free hemoglobin by optical measurement, wherein the value measured for the concentration of the analyte is corrected by the steps: (a) measure the assumed value of the sample being analyzed, (b) measure the assumed value of a reference sample free of hemoglobin, (c) measuring the incorrect value for the concentration of the analyte and (d) correcting the value obtained in step (c) by correlation with the values obtained in step (a) and (b) to obtain the corrected value for the concentration of the analyte.

The corrected value for the concentration of the analyte in step (d) of the method according to the invention is preferably determined according to the following relationship: - sample = '-sample - ^' ^ -I-mues ra "*" ^ '^ "I-reference in which C 'sample is the corrected value for the concentration of the analyte, Cmuestra is the incorrect measured value for the concentration of the analyte in the sample, F is the specific correction factor of proof, The sample is the assumed value measured in the sample and The reference is the assumed value measured in the reference sample.

The correction method according to the invention is appropriate for the methods in which the analyte is determined by optical measurement, in particular at wavelengths in which interference by the free hemoglobin present in the sample occurs. The optical measurement is particularly preferably carried out in the range of 500-750 nm.

The method according to the invention is suitable for the determination of any of the samples in which free hemoglobin is present. Examples of such samples are hemolytic serum or plasma samples or samples which contain a blood substitute. Examples of blood substitutes which fall under the term "free hemoglobin" within the meaning of the present invention are the modified or crosslinked, polymerized derivatives of hemoglobins, in particular human hemoglobin or bovine hemoglobin, as well as recombinantly produced hemoglobin. .

In a preferred embodiment of the method according to the invention, the total protein content of the sample is determined. This determination is preferably carried out according to the Biuret method. In a further particularly preferred embodiment of the present invention the iron content of the sample is determined. This determination of the iron content is preferably carried out according to the method with ferrozine. Still a further particularly preferred embodiment is determining the albumin content of a sample. This determination of the albumin content is preferably carried out according to the bromocresol-green or bromocresol-purple method. The measurement procedure for the determination of these parameters in which no simple method of eliminating interference in the determination of hemoglobin-containing samples is previously known can surprisingly be simplified by the method according to the invention.

A further feature of the method according to the invention is that it is even possible to easily measure icteric samples with a high bilirubin content to at least 20 mg / dl. An interference by the lipaeic samples can be eliminated by using a liquid reagent that makes transparent the submerged objects in it which, for example, is added to the reagent.

A serum or plasma sample and in particular a human serum or plasma sample is preferably used as the sample in the method according to the invention. Serum or plasma samples from clinically healthy test persons are advantageously used as reference samples. In particular, it is preferable to use a hemoglobin-free plasma or serum pool from clinically healthy test subjects.

A particular advantage of the method according to the invention is that it can be carried out with an automated analyzer, for example, in a Mannheim / Hitachi analyzer 704 or 717 from Boehringer. Due to the simple formula of ~ mathematical correction, the automated analyzer can be programmed in such a way that the output is already the corrected value for the concentration of the analyte and a subsequent mathematical correction is no longer necessary.

An important parameter for the correction of the measured concentration of the analyte is the specific F correction factor of the test. This correction factor F is preferably determined by a method which comprises the steps: (a) prepare a series of at least three samples with the same analyte content of which at least one of the samples does not contain hemoglobin and at least two of the samples contain different concentrations of free hemoglobin, (b) measurement of the assumed value of each sample in which the increase in the assumed value of the sample caused by the presence of hemoglobin is determined compared to the free sample of hemoglobin, (c) measuring the incorrect concentration of the analyte in each sample in which the alteration of the measured value caused by the presence of hemoglobin is determined compared to the reference sample free of hemoglobin and (d) the alteration of the measured value determined in step (c) correlates with the increase of the assumed value of the sample determined in step (b) to obtain the specific correction factor of the test.

When determining the correction factor F it is preferable to prepare a series of samples of which at least 5 and for example 10 of the samples contain different concentrations of free hemoglobin. The concentrations of free hemoglobin for example are varied for the series of samples in the range of 0 mg / dl to at least 1000 mg / dl.

For a particular sample of the series containing free hemoglobin, a correction factor F "specific to the sample is determined according to the following relationship: F1 =? C:? E1 in which:? C is the amount of the alteration of the measured value compared to the reference sample caused by the presence of free hemoglobin for each sample and ? E1 is the increase of the assumed value of the sample compared to the reference sample caused by the presence of free hemoglobin for each sample.

The correction factor F specific for the test can be determined by calculating the means of the correction factors F 'determined for the respective samples. In this way it is possible to determine a correction factor F of 0.332 for the albumin test by the bromocresol-green method, a correction factor F of 0.290 for the iron test by the ferrozine method and a correction factor of 0.115 for the total protein test by the Biuret method. When these factors are used, an excellent recovery ratio of the analyte is found in the samples that contain hemoglobin.

In addition, the invention is clarified by the following examples: General Methods: 1. Determination of the correction factor (see also examples 1-3): From a pool of serum or plasma from clinically healthy test persons, 11 samples are added with different amounts of haemosylate, hemoglobin or a compound similar to Hb in such a way that, in a constant analyte content, a series of concentrations of Hb the lowest sample is formed of which (= reference) does not contain Hb and the highest sample of which contains at least 1000 mg / dl of Hb. All samples from this series are measured with the respective test to obtain an altered value of the analyte compared to the reference value for each sample depending on its Hb content.

For each sample is the increase in the assumed value of the sample determined? The one caused by Hb compared to the assumed value of the Hb free sample (= reference): ? El = klmuestra - "Referral - In addition, the amount of alteration Δ C of the analyte value caused by Hb compared to the measured value of the analyte in the reference is determined for each sample: ? C = sample; - ^ -ref erence • When the interference component C is divided by the value of the increment of the assumed value of the sample, a correction factor is obtained for each sample F 'sample =? C:? El.

The mean correction factor F is then calculated from the 10 individual factors of the series of Hb concentrations obtained in this way. This factor is a fixed parameter, which only has to be determined once for albumin, iron and total protein and which is then, however, constant for the respective test. 2. Calculation of the corrected value of the analyte (see also examples 4-8): The corrected value and hence the value of the analyte without interference of the respective sample C'sample is determined by the mathematical correction of the measured value of the analyte of the sample CmuesCra by the interference component? C. sample = ^ -sample "? C C sample = '-sample - -t1'? Jl sample = sample - 'v -kl sample ~ klref erencia) sample = ^ -sample - i"' ^ sample + ^ '"- ^ reference • As described above, the reference is an Hb-free serum or plasma bead from clinically healthy test subjects. For the methods, the influence of the range of variation of the assumed values measured from the sample of several patient samples is negligible due to the relationship between the assumed value of the sample and the measured signal. Even icteric samples with a bilirubin content of at least 20 mg / dl do not interfere. Interference by lipemic samples can be eliminated by using a liquid reagent that makes transparent the submerged objects in it, which for example is added to the reagent. Icteric and lipemic samples are prepared by known addition of human serum with bilirubin and Intralipid® similarly by Glick (Clin.Chem./32/3, 470-475 (1986)).

In an analyzer, such as for example the Boehringer Mannheim / Hitachi 704 or 717 instruments, which automatically measures the calculation of the uninterfered value of the analyte can be programmed in such a way that only the corrected values are printed and no longer necessary. subsequent mathematical correction. This programming is for example carried out for albumin on a Mannheim / Hitachi 704 instrument by Boehringer as follows: 1. Parameter program: chemical parameters: 2. Monitor: calibration monitor (1): for test 1 enter 0 for absorbance Sl and 100,000 for K. 3. The corrected value of the analyte is calculated by the calculated test (see manual of the Mannheim / Hitachi 704 of Boehringer): Test calculated (test 2) (test 1) COnCent raCÍnreferenc? A Test 2 is the determination of the concentration of the incorrect measured value of the analyte, Test 1 is the determination of the absorbance of the assumed value of the sample, F is the factor determined for albumin, iron or total protein to correct the interference by Hb Concentrationreference = • The reference and is entered as a concentration.

Example 1 Determination of the correction factor for the determination of albumin according to the bromocresol-green method.

The determination is carried out at 37 ° C on a Mannheim / Hitachi 704 analyzer from Boehringer using analysis code 2-15-23. The following reagents are used: Reagent 1: 75 mmoles / l of succinate buffer, pH 4.2 Reagent 2: 75 mmoles / l of succinate buffer, pH 4.2; 0.3 mmoles / l bromocresol-green.

The test procedure is as follows: 350 μl of reagent 1 is added to 4 μl of sample and after the determination of the assumed value of the sample, 350 μl of reagent 2 is added. Then the analyte is determined after a period of 2 hours. minutes A main wavelength of 600 nm and a secondary wavelength of 700 nm are used for the measurement.

The result of this determination is shown in Table 1. The value for the specific test correction factor is determined as 0.332.

Example 2 Determination of the correction factor for the determination of iron according to the method with ferrizine.

The determination is carried out at 37 ° C on a Mannheim / Hitachi 717 analyzer from Boehringer using analysis code 2-24-30. The following reagents are used: Reagent 1: 150 mmoles / l of Na-acetate buffer, pH 5.0; 4 mmoles / l of guanidinium chloride; 100 mmol / l of thiourea; Detergent; Reagent 2: 150 mmoles / l of ascorbic acid, 50 mmoles / l of ferrozin.

The test procedure is as follows: 250 μl of reagent 1 is added to 20 μl of sample and after the determination of the assumed value of the sample 50 μl of reagent 2 is added. Then the analyte is determined after a period of 1 μl. minute.

A main wavelength of 546 nm and a secondary wavelength of 700 nm are used for the measurement.

The result of this experiment is shown in table 2. The value for the specific correction factor of the test is determined as 0.290.

Example 3 Determination of the correction factor for the determination of total protein according to the Biuret method.

The determination is carried out at 37 ° C on a Mannheim / Hitachi 717 analyzer from Boehringer using the analysis code 2-24-50. The following reagents are used: Reagent 1: 200 mmol / l NaOH; 32 mmoles / l of K-Na-tartrate; Reagent 2: 200 mmoles / l of NaOH; 32 mmoles / l of K-Na-tartrate; 30.5 mmol / l of Kl; 12.15 mmoles / l of copper sulfate.

The test procedure is as follows: 250 μl of reagent 1 is added to 7 μl of sample and after the determination of the assumed value of the sample, 250 μl of reagent 2 is added. Then the analyte is determined after a period of 5 hours. minutes A main wavelength of 546 nm and a secondary wavelength of 700 nm are used for the measurement.

The result of this experiment is shown in Table 3. The value for the specific correction factor of the test is determined as 0.115.

Example 4 The use of the correction formula for the determination of albumin.

The correction factor determined in example 1 is used to determine hemoglobin-containing samples which are obtained by known addition with blood substitutes.

The test procedure is as described in example 1.

The formula for calculating the corrected concentration of the analyte in the sample is as follows: C sample = sample - r * k l sample + * '^ l ref erence = sample - F The sample + 0. 3 g / 1.

The result of this experiment is shown in Table 4. It can be seen that a recovery ratio of 100 ± 1% is achieved by the correction.

Example 5 Using the correction formula for iron determination The iron is determined according to the ferrozine method using the correction factor determined in example 2 in samples containing hemoglobin which are obtained by known addition with hemolysate.

The test procedure is as described in example 2.

The formula to calculate the corrected analytical value is as follows: C sample = sample - ^ '-t- sample + ^ "" J- ref erence = sample - F He shows + 2. 9 μg / dl.

The result of this experiment is shown in Table 5. It can be seen that an excellent iron recovery is achieved which, with the exception of a single value, is in the range of 100% + 2.5%.

Example 6 Use of the correction formula for the determination of total protein The correction factor determined in example 3 is used to determine the total protein according to the Biuret method in samples containing hemoglobin, which are obtained by known addition with blood substances. The experiment is carried out as described in example 3.

The formula to calculate the corrected analytical value is as follows: C sample = ^ -sample - ^ "" Show + ^ '^ Iref erencia = mUestra - F ElmUestra + 0. 6 g / 1.

The result of this experiment is shown in Table 6. It can be seen that the recovery for the total protein is in some cases in the range of 100% + 1%.

Example 7 Determination of albumin in icteric samples.

The test specific correction factor determined in Example 1 is used to determine the albumin according to the bromocresol-green method in the icteric samples which are obtained by known addition with bilirubin.

The test procedure is as described in example 1. The formula for calculating the corrected analytical value is as described in example 4.

The result of this experiment is shown in Table 7. It can be seen that the use of the correction formula does not lead to a worsening of the recovery.

Example 8 Determination of iron in lipemic samples The test specific correction factor determined in example 2 is used to determine iron according to the ferrozine method in lipemic samples which are obtained by known addition with Intralipid®.

The test procedure is as described in example 2. The formula for calculating the corrected analytical value is as given in example 5.

The result of the experiment is shown in Table 8. It can be seen that the use of the correction formula does not lead to a worsening of the recovery in the lipemic samples.

Table 1 Table 2 r \ - > Table 3 ro Table 4 ro cx > Table 5 ro Table 6 J o Table 7 Table 8 ro It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property.

Claims

1. The method for the determination of an analyte in a sample containing free hemoglobin by optical measurement, characterized in that the value measured for the concentration of the analyte is corrected by the steps: (a) measure the assumed value of the sample being analyzed, (b) measure the assumed value of a reference sample free of hemoglobin, (c) measuring the incorrect value for the concentration of the analyte and (d) correcting the value obtained in step (c) by correlation with the values obtained in steps (a) and (b) to obtain the corrected value for the concentration of the analyte, where the corrected value for the concentration of the analyte is calculated according to the following relationship: sample = ^ -sample - ^ '"-" - sample +' "-1-reference in which C sample is the corrected value for the concentration of the analyte, Sample is the incorrect measured value for the concentration of the analyte in the sample, F is a specific correction factor of proof, The sample is the assumed value measured in the sample and the reference is the value measured in the reference sample.

2. The method according to claim 1, characterized in that the determination of the analyte is carried out by an optical measurement in the range of 600-700 nm.

3. The method according to one of claims 1-2, characterized in that a sample is determined which contains a blood substitute.

4. The method according to one of claims 1-3, characterized in that the total protein content of the sample is determined.

5. The method according to claim 4, characterized in that the determination of the total protein content is carried out according to the Biuret method.

6. The method according to one of claims 1-3, characterized in that the iron content is determined.

7. The method according to claim 6, characterized in that the iron content is determined according to the ferrozine method.

8. The method according to one of claims 1-3, characterized in that the albumin content of the sample is determined.

9. The method according to claim 8, characterized in that the content of albumin is determined according to the method with bromocresol-green or bromocresol-purple.

10. The method according to one of claims 1-9, characterized in that in the determination of the lipemic samples a liquid reagent is added which makes the submerged objects transparent therein.

11. The method according to one of claims 1-10, characterized in that the determination is carried out in a serum or plasma sample.

12. The method according to one of claims 1-11, characterized in that serum or plasma samples from clinically healthy test persons are used as a reference sample.

13. The method according to one of claims 1-12, characterized in that the method is carried out in an automated analyzer.

14. The method according to claim 13, characterized in that the automated analyzer is programmed in such a way that the corrected value for the concentration of the analyte is printed immediately.

15. The method according to claim 1, characterized in that the determination of the correction factor F specific test comprises the steps: (a) prepare a series of at least three samples with the same analyte content, of which at least one of the samples does not contain hemoglobin and at least two of the samples contain different concentrations of free hemoglobin, (b) measure the assumed value of each sample to determine the increase in the assumed value of the sample caused by the presence of hemoglobin compared to the free sample of hemoglobin, (c) measuring the incorrect concentration of the analyte in each sample to determine the alteration of the measured value caused by the presence of hemoglobin compared to the reference sample free of hemoglobin and (d) the alteration of the measured value determined in step (c) is correlated with the increment of the assumed value of the sample determined in step (b) to obtain the specific correction factor of the test.

16. The method according to claim 15, characterized in that a series of samples is prepared, of which at least 5 of the samples contain different concentrations of free hemoglobin.

17. The method according to claim 15 or 16, characterized in that a series of samples is prepared, of which the concentration varies from 0 mg / dl to at least 1000 mg / dl of free hemoglobin.

18. The method according to one of claims 15-17, characterized in that a sample-specific correction factor F1 is determined for a sample containing free hemoglobin according to the following relationship: F '=? C:? The one in which:? C is the amount of alteration of the measured value compared to the reference sample caused by the presence of free hemoglobin for an individual sample and ? The is the increment of the assumed value of the sample compared to the reference sample caused by the presence of free hemoglobin for an individual sample, and the specific correction factor F of the test is determined by calculating the means of the correction factors F 'determined for the respective individual samples.