PROCEDURE FOR THE ELIMINATION OF HEMOGLOBIN INTERFERENCES IN THE ANALYSIS OF MEDICAL SAMPLES
DESCRIPTION OF THE INVENTION 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 means of an optical biochromatic measurement at a primary and a secondary wavelength. In particular, this method is suitable for the determination of parameters of ammonia, creatinine kinase and its isoenzymes and hydrogenase lactate and its enzymes in a medical sample, for example a serum or plasma sample. It is generally known that hemolysis interferes with the determination of many analytes in some cases to a considerable degree. In order to obtain non-falsified measured values in any way, various methods for eliminating interference by hemolysis have been previously published. One such method is that during a measurement in automatic analyzers a second wavelength (secondary wavelength) is used in addition to the first wavelength (primary wavelength) by means of which it can be eliminated or at least minimized the influence of interfering substances such as hemoglobin, bilirubin and lipemia. A requirement for this is that the substance that is going
REF .: 28210 to measure absorb as little as possible in the wavelength, but on the contrary the interfering substance absorbs as much as possible at almost the same level as in the main wavelength (Praxis echni: Photometer für die Árztliche Praxis, Deutscher Arzteverlag 1977, pages 41-42). In the writing DAY (Boehringer Mannheim) no. 70 (1985) it is mentioned that the secondary wavelength should be as similar as possible to the main wavelength, since usually the interfering substance presents extinctions similar to the main and secondary wavelengths. In Clin.Chem. 25/6, 951-959 (1979) is s * á¡É < ? a_-recommends selecting the secondary wavelength so that it is close to the minimum absorption of the chromogen and close to the absorption maximum of the interfering substance. In this regard, a secondary wavelength of 280 nm is recommended for the determination of glucose (main wavelength of 340 nm), since interfering substances absorb here in a manner similar to that of 340 nm. In Eur. J. Clin.Biochem 31/9, 595-601 (1993), on the other hand, it is considered critical to measure UV tests with a secondary wavelength of 380 nm, since in this case the transformation of Hb-02 into Meth-Hb leads to spectral modifications at 380 nm and with this to measurement errors. For tests that are based on measuring the reduction or increase of NAD (P) H, wavelengths are recommended, which in turn are in the so-called Soret region, such as 475 nm. All the procedures described above refer to the elimination of disturbances of erroneous measurements caused by hemolysis. With the preparation of blood substitutes based on hemoglobin, the problem of the elimination of interferences by natural or synthetic hemoglobin or Hb analogues is presented, more sharply than before. These interferences occur on the one hand also in non-hemolytic material, and on the other hand also to a higher degree than in natural hemolysis, since in blood substitute therapy the Hb content in blood serum or plasma can be of up to 2000 mg / dl. It was further determined that in the measurement certain analytes such as ammonia, creatinine kinase and its isoenzymes as well as lactate dihydrogenase and its isoenzymes it is not easily possible to obtain an adequate elimination of the hemoglobin interference when using a wavelength secondary level of 475 nm or greater, for example to 480, 505, 600, 660 or 700 n ..- since these parameters are of essential importance in the context of cardiovascular and emergency diagnosis as well as for the diagnosis of patients treated with substitutes of blood, the object of the present invention is to provide a simple method for eliminating interferences caused by natural hemoglobin or by blood substitutes based on synthetic hemoglobin or hemoglobin-like compounds, in particular when measuring the aforementioned analytes . The object of the invention is achieved by means of a method for the determination of an analyte in a sample containing free hemoglobin by optical bichromatic measurements at a main or secondary wavelength, using a secondary wavelength greater than 475 nm , in which the hemoglobin absorption bands are found. The preferred secondary wavelengths for the method according to the invention are in the range of 546 ± 10 nm, in particular 546 ± 5 nm, as well as in the range of 570 ± 10 nm in particular 570 ± 5 nm- The wavelengths of 546 and 570 are most preferred. The selection of the wavelengths according to the invention as secondary wavelengths was surprising since the highest interferences by hemoglobin are obtained at the secondary wavelength of 405 nm known from the state of the art (for example calibration of the instrument for the Boehringer Mannheim / Hitachi 717 analyzer according to the instructions of the team for the reagent to determine the creatinine kinase, order No. 1 273 248, Diagnostic catalog Boehringer Mannheim Diagnostica) to which an absorption through the hemoglobin. Also in the aforementioned publication Eur.J. Clin. Chem. Clin. Biochem indicates that a secondary wavelength, which lies beyond the soret region (the main absorption bands of hemoglobin), should be used for the elimination of hemoglobin interference, so that in any case it would be obvious select as wavelengths those wavelengths, where no band of hemoglobin absorption is found. The method for eliminating the interference according to the invention is suitable for the methods in which the analyte is determined by means of optical measurement, in particular by means of optical measurements at a main wavelength in the UV range. The method is preferably performed for tests that are based on a measurement of the increase or decrease in the concentration of NADH or NADPH in a sample. In this case, a main measured wavelength in the range of 340 + 10 nm is preferably used. The method according to the invention is suitable for the determination of certain samples in which free hemoglobin is found. Examples of these samples are the hemolytic samples of serum or plasma or samples that contain a blood substitute. Examples of blood substitutes which, according to the meaning of the present invention, fall within the concept of "free hemoglobin" without derivatives of derivatized, polymerized, modified or crosslinked hemoglobins, in particular of human hemoglobin or bovine hemoglobin, such as, for example, hemoglobin DCL (Diaspirin reticulated hemoglobin), as well as hemoglobin prepared recombinantly. In a preferred embodiment, the method according to the invention determines the content of an analyte selected from the group consisting of ammonia, creatinine kinase and its insoenzymes and lactate dihydrogenase and its isoenzymes. The determination of ammonia by the process according to the invention is preferably carried out according to the enzymatic UV method (Da Fonseca-Wollheim F., Z.Klin, Chem.Biochem, 11 (1973) 421). * The determination of creatinine kinase (CK) is preferably carried out according to the "Optimized standard method" of the German Society of Clinical Chemistry (J.Clin.Chem.Clin.Biochem.15 (1977), 249). The determination of the creatinine kinase isoenzymes CK-MB is preferably carried out according to the immunological UV method (Würzburg U. et al., Klin. schr. 54 (1976), 357). The determination of lactate dihydrogenase (LDH) or of lactate dihydrogenase isozymes (HBDH) (l-hydroxybutyrate dihydrogenase) is preferably carried out in accordance with the "Optimized standard method" of the German Society of Clinical Chemistry (Z.Klin. Chem. Klin.Biochem 8 (1970), 658 and 10 (1972), 182). As a sample, a sample of serum or plasma, in particular a sample of human serum or plasma, is used in the process according to the invention. A special advantage of the process according to the invention is that it can be carried out in an automatic analyzer, for example a Boehringer Mannheim / Hitachi 704-711 analysis apparatus. In such analyzers, especially preferred secondary wavelengths of 546 or 570 nm can be easily adjusted. The invention will now be clarified by means of the following examples: General methods A part of a serum tank was mixed with a solution containing hemoglobin in such a way that a hemoglobin content of 2000 μg / dl was achieved. Another part of the serum tank of the same size was mixed with the equivalent amount of a Na-Cl solution (154 mmol / 1). Both parts were mixed together in a different proportion in such a way that a series of Hb concentrations of 11 samples were formed, there being one sample that did not contain Hb and the largest sample contained 2000 mg / dl. Example 1 Determination of ammonia The determination was carried out on a Boehringer Mannheim / Hitachi 717 analyzer. The following reagents were used: Reagent 1: 150 mmol / 1 of triethanolamine buffer, pH 8.6; 15 mmol / 1 < > - ketoglutarate; 1.5 mmol / 1 ADP Reagent 2: 150 mmol / 1 triethanolamine buffer; pH 8.6; 15 mmol / 1 ^ -ketoglutarate; 1.5 mmol / 1 ADP; 0.31 mmol / 1 NADPH; > 24 U / ml of glutamate desidrogenase (GLDH) The test was carried out as follows: 20 μl of the sample were added with 200 μl of reagent 1 and after 5 minutes 50 μl of reagent 2. The analyte was determined. I do it after a period of another 40 seconds. For the measurement, main wavelengths of 340 nm and secondary wavelengths of 05 nm, 480 nm, 505 nm, 600 nm, 660 nm and 700 nm (comparative) as well as 546 nm and 570 nm (invention) were used. The results of that determination are shown in Table 1. It should be recognized that using the wavelengths according to the invention of 546 or 570 nm resulted in a clearly improved recovery than with other wavelengths. Example 2 Determination of creatinine kinase The determination was made on a Boehringer Mannheim / Hitachi 717 analyzer. The following reagents were used: Reagent 1: 110 mmol / 1 imidazole buffer; pH 6.7; 20.5 mmol / 1 glucose, 2.05? Ranol / 1 EDTA; 2.5 MMOL / 1 ADP; 6.1 mmol / 1 AMP; 12 μmol / 1 diadenosinepentaphosphate; 2.5 mmol / 1 NADP; 25 mmol / 1 N-acetylcysteine; > 3.1 U / ml hexokinase (HK); > 1.8 U / ml glucose-6-phosphate dehydrogenase (G6P-DH) Reagent 2: 25 mmol / 1 imidazole buffer; pH 7.5; 20.5 mmol / 1 glucose; 2.05 mmol / 1 EDTA; 61 mmol / 1 Mg2 *; 184 mmol / 1 of creatinine phosphate The test was carried out as follows: To a sample of 7 μl was added 250 μl of reagent 1 and after 5 minutes 50 μl of reagent 2. The determination of the analyte was made after a period of another 2 minutes. For the measurement a main wavelength of 340 nm and secondary wavelengths of 405 nm, 480 nm, 505 nm, 600 nm, 660 nm and 700 nm (comparative) as well as 546 nm and 570 nm were used.
(invention). The results of that determination are shown in Table 2. It should be recognized that using the wavelengths according to the invention of 546 or 570 nm resulted in a clearly improved recovery than with other wavelengths. Example 3 Determination of CK-MB creatinine kinase isoenzymes The determination was made on an analyzer
Boehringer Mannheim / Hitachi 717. The following reagents were used: Reagent I: 110 mmol / 1 imidazole buffer; pH 6.7; 21 mmol / 1 glucose, 11 mmol / 1 Mg2 +; 2.1 mmol / l EDTA; 2.4 mmol / l ADP; 6.0 mmol / 1 AMP; 12 μmol / 1 diadenosinepentaphosphate; 2.4 mmol / 1 NADP; 24 mmol / 1 N-acetylcysteine; > 3.0 U / ml HK; > 1.8 U / ml GßP-DH; antibodies, inhibitory capacity against CK-M up to 2000 U / 1 Reagent 2: 110 mmol / 1 imidazole buffer; pH 6.7; 21 mmol / 1 glucose; 2.1 mmol / 1 EDTA; 11 mmol / 1 Mg2 *; 186 mmol / 1 of creatinine phosphate The test was carried out as follows: To a 12 μl sample, 250 μl of reagent 1 was added and after 50 minutes, 50 μl of reagent 2 was added. The analyte was determined after a period of another 3 minutes. For the measurement, a main wavelength of 340 nm and secondary wavelengths of 405 nm, 480 nm, 505 nm, 600 nm, 660 nm and 700 nm (comparative) as well as 546 nm and 570 nm (invention) were used. The results of that determination are shown in Table 2. It should be recognized that using the wavelengths according to the invention of 546 or 570 nm resulted in a clearly improved recovery than with other wavelengths. Example 4 Determination of lactate dihydrogenase The determination was made in an analyzer
Boehringer Mannheim / Hitachi 717. The following reagents were used: Reagent 1: 68 mmol / 1 phosphate buffer; pH 7.5; > .0.73 mmol / 1 pyruvate Reagent 2: > 1.1 mmol / l NADH The test was carried out as follows: To a 5 μl sample, 250 μl of reagent 1 was added and after 50 minutes, 50 μl of reagent 2. The analyte was determined after a period of another 60 seconds. For the measurement, a main wavelength of 340 nm and secondary wavelengths of 405 nm, 480 nm, 505 nm, 600 nm, 660 nm and 700 nm (comparative) as well as 546 nm and 570 nm (invention) were used. The results of that determination are shown in Table 2. It should be recognized that using the wavelengths according to the invention of 546 or 570 nm resulted in a clearly improved recovery than with other wavelengths. Example 5 Determination of isoenzymes of LDH HBDH The determination was carried out on a Boehringer Mannheim / Hitachi 717 analyzer. The following reagents were used: Reagent 1: 68 mmol / 1 phosphate buffer; pH 7.5; 3.7 mmol / 1 * -oxobutyrate Reagent 2: > 1.1 mmol / 1 NADH The test was carried out as follows: To a 5 μl sample was added 250 μl of reagent 1 and after 5 minutes 50 μl of reagent 2. The determination of the analyte was carried out after a period of another 60 seconds. For the measurement a main wavelength of 340 nm and secondary wavelengths of 405 nm, 480 nm, 505 nm, 600 nm, 660 nm and 700 nm (comparative) as well as 546 nm and 570 nm were used.
(invention). The results of that determination are shown in Table 2. It should be recognized that using the wavelengths according to the invention of 546 or 570 nm resulted in a clearly improved recovery than with other wavelengths.
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It is noted that in relation to this date the best method known by 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, it is claimed as property contained in the following: