NZ331491A

NZ331491A - Process to eliminate haemoglobin errors when analysing medical samples using a main and a secondary measuring wavelength

Info

Publication number: NZ331491A
Application number: NZ331491A
Authority: NZ
Inventors: Ralph Weisheit; Elke Pfitschler
Original assignee: Boehringer Mannheim Gmbh
Priority date: 1996-05-31
Filing date: 1997-05-30
Publication date: 1999-04-29
Also published as: PL330220A1; JPH11510902A; DK0906570T3; DE19622090A1; TW407202B; AU3093597A; WO1997045733A1; EP0906570A1; CN1216614A; ES2154463T3; CA2255851A1; HUP9903344A2; EP0906570B1; IL127275A0; ATE198670T1; KR20000010767A; CZ389598A3; DE59702900D1

Abstract

The present invention relates to a process for determining an analyte in a sample containing free haemoglobin by optical bichromatic measurement for a main and a secondary measuring wave length. A secondary measuring wave length of above 475 nm is used containing absorption bands of haemoglobin.

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number 331491 New Zealand No. 331491 International No. PCT/EP97/02835 TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates: 31.05.1996; Complete Specification Filed: 30.05.1997 Classification.(6) G01N33/52,70,72; C12Q1/00.12 Publication date: 29 April 1999 Journal No.: 1439 Title of Invention: Process to eliminate haemoglobin errors when analysing medical samples Name, address and nationality of applicant(s) as in international application form: BOEHRINGER MANNHEIM GMBH, Sandhofer Strasse 112-132, D-68305, Mannheim, Germany NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION 33149 1 I - Method for the elimination of haemoglobin interferences in the analysis of medical samples Description The invention concerns a method for the determination of an analyte in a sample containing free haemoglobin in which the determination is carried out by an optical bichromatic measurement at a main and a secondary wavelength. In particular this method is suitable for the determination of the parameters ammonia, creatine kinase and isoenzymes thereof and lactate dehydrogenase and isoenzymes thereof in a medical sample e.g. a serum or plasma sample. It is generally known that haemolysis interferes with the determination of many analytes in some cases to a considerable extent. In order to nevertheless obtain measured values which are unfalsified various methods have been published in the past for eliminating haemolysis interference. One of these methods is that when measuring in automated analyzers a second wavelength (secondary wavelength) is used in addition to the first wavelength (primary wavelength) by means of which the interfering influence of interfering substances such as haemoglobin, bilirubin and lipaemia can be eliminated or at least minimized. One requirement for this is that the substance to be measured absorbs as little as possible at the secondary wavelength but that the interfering substance absorbs at the same level as possible as at the main wavelength 3314: - 2 - ("Praxistechnik: Photometer fiir die Arztliche Praxis, Deutscher Xrzteverlag" 1977, pages 41-42). In the DIA letter (Boehringer Mannheim) No. 70 (1985) it is mentioned that the secondary wavelength should be as close as possible to the main wavelength since as a rule the interfering substance then has similar absorbances at the main and secondary wavelengths. In Clin. Chem. 25/6, 951-959 (1979) it is pointed out that the secondary wavelength should be selected so that it is near to the absorption minimum of the chromogen and near to the absorption maximum of the interfering substance. In this connection a secondary wavelength of 380 nm is recommended for the determination of glucose (main wavelength 34 0 nm) since here the absorbance of the interfering substances is similar to that at 340 nm. In contrast in the Eur. J. Clin. Chem. Clin. Biochem. 31/9, 595-601 (1993) it is regarded as critical to measure UV tests at a secondary wavelength of 380 nm since in this case the conversion of Hb-02 into Meth-Hb leads to spectral changes at 380 nm and thus to errors in the measurements. Therefore a secondary wavelength is recommended for tests which are based on the measurement of a decrease or increase in NAD(P)H which lies behind the so-called Soret region such as e.g. 475 nm. All previously described methods relate to the elimination of interference in erroneous measurements caused by haemolysis. The availability of blood substitutes based on haemoglobin makes the issue of removing interferences by native or synthetic haemoglobin or compounds analogous to Hb much more acute - 3 - 3314 than hitherto. Such interferences then on the one hand also occur in non-haemolytic sample material and on the other hand also to a much greater extent than in native haemolysis since the haemoglobin content of blood serum or plasma can be up to 2000 mg/dl in blood substitute therapy. In addition it was found that when measuring certain analytes such as ammonia, creatine kinase and isoenzymes thereof as well as lactate dehydrogenase and isoenzymes thereof it is not easily possible to achieve an adequate elimination of haemoglobin interference by using a secondary wavelength of 475 nm or higher e.g. at 480, 505, 600, 660 or 700 nm. Since these parameters are of essential importance in the context of cardiovascular and emergency diagnostics as well as for the diagnosis of patients treated with blood substitutes, the object of the present invention was to provide a simple method for eliminating interferences which are caused by native haemoglobin or by blood substitutes based on synthetic haemoglobin or compounds similar to haemoglobin, in particular when measuring the above-mentioned analytes. The object of the invention is achieved by a method for the determination of an analyte in a sample containing free haemoglobin by optical bichromatic measurement at a main and a secondary wavelength wherein a secondary wavelength above 475 nm is used in which the absorption bands of haemoglobin are located. 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 and in particular 570 ± 5 nm. The wavelengths 546 - 4 - 3314 and 570 nm are most preferred. The selection of the wavelengths according to the invention as secondary wavelengths was surprising since the greatest interferences by haemoglobin are obtained at the secondary wavelength of 405 nm known from the state of the art (for example instrument settings for the Boehringer Mannheim/Hitachi 717-analyser according to the instructions of the kit insert for the reagent to determine creatine kinase, order No. 1 273 248, Boehringer Mannheim Diagnostica Catalogue 1997) at which haemoglobin also absorbs. Furthermore in the aforementioned publication Eur. J. Clin. Chem. Clin. Biochem. it is pointed out that a secondary wavelength should be used to eliminate interference by haemoglobin which lies beyond the Soret region (the main absorption band of haemoglobin) so that it would at most have been obvious to use those wavelengths as a secondary wavelength where there are no absorption bands at all of haemoglobin. The method for eliminating interference according to the invention is suitable for methods in which the analyte is determined by optical measurement in particular by optical measurement at a main wavelength in the UV range. The method is particularly preferably carried out for tests which are based on a measurement of the increase or decrease of the concentration of NADH or NADPH in the sample. In this case one preferably uses a main wavelength in the range of 34 0 ± 10 nm. amended page 33 14 4a The method according to the invention is suitable for the determination of any samples in which free haemoglobin is present. Examples of such samples are haemolytic serum or plasma samples or samples which contain a blood substitute. Examples of blood substitutes which fall under the term "free haemoglobin" within the sense of the present invention are amended page - 5 - 33 14 derivatized, polymerized, modified or cross-linked derivatives of haemoglobins in particular of human haemoglobin or bovine haemoglobin such as DCL haemoglobin (diaspirin cross-linked haemoglobin) and recombinantly produced haemoglobin. In a preferred embodiment of the method according to the invention the content of an analyte selected from the group comprising ammonia, creatine kinase and isoenzymes thereof and lactate dehydrogenase and isoenzymes thereof is determined. The determination of ammonia by the method according to the invention is preferably carried out according to the enzymatic UV method (Da Fonseca-Wollheim F., Z. Klin. Chem. Klin. Biochem. 11 (1973) 421). Creatine kinase (CK) is preferably determined according to the "optimized standard method" of the German Society for Clinical Chemistry (J. Clin. Chem. Clin. Biochem. 15 (1577) , 249). The creatine kinase isoenzyme CK-MB is preferably determined by the immunological UV method (Wiirzburg U. et al., Klin. Wschr. 54 (1976), 357). The determination of lactate dehydrogenase (LDH) or of the lactate dehydrogenase isoenzyme (HBDH) (1-hydroxy-butyrate dehydrogenase) is preferably carried out according to the "optimized standard method" of the German Society for Clinical Chemistry (Z. Klin. Chem. Klin. Biochem. 8 (1970), 658 and 10 (1972), 182). A serum or plasma sample is preferably used as the sample in the method according to the invention in particular a human serum or plasma sample. - 6 - 33 1491 A particular advantage of the method according to the invention is that it can be carried out in an automated analyzer such as a Boehringer Kannheim/Hitachi 704 or 717 analyzer. In such analyzers it is easily possible to set the particularly preferred secondary wavelengths of 546 or 570 nm. The invention is further elucidated by the following examples. General methods A solution containing haemoglobin was added to one portion of a serum pool such that a haemoglobin content of 2000 mg/dl was reached. Another equal portion of the serum pool was admixed with an equivalent amount of a NaCl solution (154 mmol/1)„ Both portions were subsequently mixed together in different ratios in such a way that a Kb concentration series of 11 samples was formed, whereby one sample contained no Hb and the highest sample contained 2000 mg/dl Hb. - 7 - 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 iamol/1 triethanolamine buffer, pH 8.6; 15 mmol/1 a-ketoglutarate; 1.5 mmol/1 ADP Reagent 2: 150 mmol/1 triethanolamine buffer; pH 8.5; 15 mmol/1 a-ketoglutarate; 1.5 mmol/1 ADP; 0.31 mmol/1 NADFH; > 24 U/ml glutamate dehydrogenase (GLDH) The test procedure was as follows: 200 /u 1 reagent l and after 5 min 50 jul reagent 2 were added to 2 0 ;ul sample. The analyte was determined after a period of a further 40 sec. A main wavelength of 340 nm and secondary wavelengths of 405 nm, 480 nm, 505 nm, 600 nut, 660 nm and 700 nm (comparison) as well as of 546 nm and 570 nm (invention) were used for the measurement. The result of this determination is shown in table 1. It can be seen that when using the measurement wavelengths of 546 and 570 nm according to the invention a considerably improved recovery was achieved than with the other wavelengths. 33 149 Example 2 Determination of creatinine kinase The determination was carried out 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 mmol/1 EDTA; 2.5 mmol/1 ADP; 6.1 mmol/1 AMP; 12 /Limol/1 diadenosine pentaphosphate; 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 creatine phosphate The test procedure was as follows: 250 /xl reagent 1 and after 5 min 50 jxl reagent 2 were added to 7 /xl sample. The analyte was determined after a period of a further 2 min. A main wavelength of 340 nm and secondary wavelengths of 405 nm, 480 nm, 505 nm, 600 nm, 660 nm and 700 nm (comparison) as well as of 546 nm and 570 nm (invention) were used for the measurement. The result of this determination is shown in table 2. It can be seen that when using the measurement wavelengths of 546 and 570 nm according to the invention a considerably improved recovery was achieved than with 33 14 - 9 - the other wavelengths. Example 3 Determination of the creatine kinase isoenzyme CK-MB The determination was carried out on a Boehringer Mannheim/Hitachi 717 analyzer. The following reagents were used: Reagent l: 110 mtiol/l imidazole buffer; pH 6.7; 21 mmol/1 glucose; 11 mmol/1 Mg2+; ZJ mmol/1 EDTA; 2.4 mmol/1 ADP; 6.0 mmol/1 AMP; 12 /imol/1 diadenosine pentaphosphate; 2.4 mmol/1 NADP; 24 mmol/1 N-acetyl cysteine; >3.0 U/ml HK; > 1.8 U/ml G6P-DH; antibody, inhibitory capacity towards CK-M up to 2000 U/l. 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 creatine phosphate The test procedure was as follows: 250 /z 1 reagent 1 and after 5 min 50 fil reagent 2 were added to 12 fxl sample. The analyte was determined after a period of a further 3 min. A main wavelength of 340 nm and secondary wavelengths of 4 05 nm, 480 nm, 505 nm, 600 nm, 660 nm and 700 nm (comparison) as well as of 546 nm and 570 nn (invention) were used for the measurement. The result of this determination is shown in table 3. It - 10 - 3314 can be seen that when using the measurement wavelengths of 546 and 570 nm according to the invention a considerably improved recovery was achieved than with the other wavelengths. Example 4 Determination of lactate dehydrogenase 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; > 0.73 mmol/1 pyruvate Reagent 2: >1.1 mmol/1 NADH The test procedure was as follows: 250 /liI reagent 1 and after 5 min 50 /xl reagent 2 were added to 5 nl sample. The analyte was determined after a period of a further 60 sec. A main wavelength of 34 0 nm and secondary wavelengths of 4 05 nm, 480 nm, 505 nm, 600 nm, 660 nm and 700 nm (comparison) as well as of 546 nm and 570 nm (invention) were used for the measurement. The result of this determination is shown in table 4. It can be seen that when using the measurement wavelengths of 546 and 570 nm according to the invention a considerably improved recovery was achieved than with the other wavelengths. - 11 - Example 5 Determination of the LDH isoenzyme 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 a-oxobutyrate Reagent 2: >1-1 mmol/1 NADH The test procedure was as follows: 250 fil reagent 1 and after 5 min 50 ^1 reagent 2 were added to 5 /xl sample. The analyte was determined after a period of a further 60 sec. A main wavelength of 340 nm and secondary wavelengths of 405 nm, 480 nm, 505 nm, 600 nm, 660 nm and 700 nm (comparison) as well as of 546 nm and 570 nm (invention) were used for the measurement. The result of this determination is shown in table 5. It can be seen that when using the measurement wavelengths of 546 and 570 nm according to the invention a considerably improved recovery was achieved than with the other wavelengths. Table 1 Sample Hb content Content at Content at Content at Content at Content at Content at Content at Content at [mg/dl] 405 nm 480 nm 505 nm 546 nm 570 nm 600 nm 660 nm 700 nm fcg/dU [ng/dl] Cng/di] kg/di] [nam fug/dl] tug/di] fug/di] 1 0 157 163 160 159 158 154 158 154 2 200 145 162 162 157 156 158 ' 157 155 3 400 140 165 163 162 158 162 158 160 4 600 133 166 162 161 153 163 162 161 5 800 145 172 171 164 158 168 166 158 6 1000 144 173 172 162 155 168 171 167 7 1200 160 171 175 167 156 175 166 164 8 14G0 ' 159 177 177 166 155 177 171 170 9 1600 176 180 178 162 152 178 178 180 10 1800 175 183 177 163 157 188 180 183 11 2000 178 189 192 172 159 199 190 187 Sample Hb content J [mg/dl] j Recovery 405 nm [%] Recovery 480 nm [%] Recovery 505 nm [%] Recovery 546 nm [%] Recovery 570 nm P/o] Recovery 600 nm [%] Recovery 660 nm [%] Recovery 700 nm [%] 1 0 100 100 100 100 100 100 100 100 2 200 92 99 101 99 99 103 99 101 3 400 89 101 102 102 100 105 100 104 4 600 85 102 101 101 97 106 103 105 5 800 92 106 107 103 100 109 105 103 6 1000 92 106 108 102 98 109 108 108 7 1200 102 105 109 105 99 114 105 10S 8 1400 101 109 111 104 98 115 108 110 9 1600 112 110 111 102 96 116 113 117 10 1800 111 112 111 103 100 122 114 119 11 2000 | 113 116 120 108 101 129 120 121 CM C*l - 13 -Table 2 Sample Hb content I Content at Content at Content at Content at Content at Content at Content at Content at [mg/dl] 405 nm 480 nm 505 nm 546 nm 570 nm 600 nm 660 nm 700 nm I [U/l] [U/l] [U/l] [U/l] [U/l] [U/l] [U/l] [U/I] 0 175 175 177 175 177 176 179 174 2 200 181 176 177 175 180 176 180 178 3 400 189 177 179 178 180 176 ■ 179 178 4 600 182 165 165 168 171 165 170 166 5 800 191 164 166 168 170 162 166 166 6 1000 185 164 164 170 172 163 166 163 7 1200 196 163 164 170 174 163 166 165 8 1400 205 163 163 170 174 160 165 164 9 1600 212 163 163 173 174 161 164 162 10 1800 214 160 163 172 177 161 165 163 11 2000 224 160 161 173 178 160 164 164 | Sample Hb content Recovery Recovery Recovery Recovery Recovery Recovery Recovery Recovery [mg/dl] 405 nm 480 nm 505 nm 546 nm 570 nm 600 nm 660 nm 700 nm [%] [%] [%] [%] [%] [%] [%] [%] 1 0 100 100 100 100 100 100 100 100 2 200 103 101 100 100 102 100 101 102 3 400 108 101 101 102 102 100 100 102 4 600 104 94 93 96 97 94 95 95 5 800 109 94 94 96 96 92 93 95 6 1000 111 94 93 97 97 93 93 94 7 1200 112 93 93 97 98 93 93 95 8 1400 117 93 92 97 98 91 92 94 9 1600 121 93 92 99 98 91 92 93 10 1800 122 91 92 98 100 91 92 94 11 2000 128 91 91 99 101 91 92 94 c*i • e • • • • - 14 -Table 3 Sample Hb content Content at Content at Content at Content at Content at Content at Content at Content at [mg/dl] 405 nm 480 nm 505 nm 546 nm 570 nm 600 nm 660 nm 700 nm [U/l] [U/l] [U/l] [U/l] [U/l] [U/l] [U/l] [U/l] 1 0 40.6 40.2 40.7 41.5 40.1 40.7 40.8 38.1 2 200 49.7 41.2 39.4 40.7 42.6 39.1 39.7 37.9 3 400 58.9 37.3 38.2 41.8 41.6 38.8 • 36.0 37.2 4 600 68.1 38.5 36.8 43.1 40.8 36.1 37.7 36.5 5 800 75.3 36.6 35.7 42.6 42.4 32.7 36.3 32.5 6 1000 82.4 34.9 35.4 44.1 43.4 30.8 32.6 36.5 7 1200 82.5 39.6 33.9 44.4 44.5 27.5 36.7 32.8 8 1400 95.0 35.5 34.5 44.8 43.0 2C.1 33.4 33.9 9 1600 99.2 37.3 35.0 44.1 44.3 25.9 31.7 29.4 10 1800 98.9 33.9 29.8 46.7 42.9 24.8 27.1 29.4 11 2000 97.1 33.2 29.1 48.1 44.1 22.4 27.9 29.9 ! Sample Hb content [mg/dl] Recovery 405 nm [%] Recovery 480 nm [%] Recovery 505 nm [%] Recovery 546 nm r%] Recovery 570 nm [%] Recovery 600 nm [%] Recovery 660 nm [%] Recovery | 700 nm [%] 1 0 100 100 100 100 100 100 100 100 2 200 122 102 97 98 106 96 97 99 3 400 145 93 94 101 104 95 88 98 4 600 168 96 90 104 102 89 92 96 5 800 185 91 88 103 106 80 89 85 6 1000 203 87 87 106 108 76 80 96 7 1200 203 98 83 107 111 68 90 86 8 1400 234 88 85 108 107 64 82 89 9 1600 244 93 86 106 110 64 78 77 10 1800 244 84 73 113 107 61 66 11 11 2000 I 239 83 71 116 110 55 68 78 | • • • • • « - 15 -Table 4 Sample Hbcontent Content at Content at Content at Content at Content at Content at Content at Content at [mg/dl] • 405 nm 480 nm 505 nm 546 nm 570 nm 600 nm 660 nm 700 nm I [U/l] [U/l] [U/l] [U/l] [U/l] [U/l] [U/l] [U/l] 1 0 196 197 199 197 199 199 199 * 11 " { 205 2 200 162 202 206 203 198 208 200 206 3 400 127 210 215 199 195 219 207 215 4 600 95 218 223 202 196 229 217 224 5 800 72 220 228 200 192 225 222 222 6 1000 45 224 226 199 193 230 226 222 7 1200 27 223 232 201 190 236 228 233 8 1400 11 237 240 203 192 243 234 236 q 1600 -13 230 238 199 190 242 233 236 10 1800 -1 234 244 198 192 245 237 238 11 2000 -15 233 246 197 191 247 238 241 I mple Hb content [mg/dl] Recovery 405 nm I [%] Recovery 480 nm [%] Recovery 505 nm [%] Recovery 546 nm [%] Recovery 570 nm [%] Recovery 600 nm [%] Recovery 660 nm [%] Recovery I 700 nm [%] 1 0 100 100 100 100 100 100 100 100 2 200 83 103 104 103 99 104 101 100 3 400 65 107 108 101 98 110 104 105 4 600 48 111 112 103 98 115 109 109 5 800 37 112 115 102 96 113 112 108 6 1000 23 114 114 101 97 116 114 108 7 1200 14 113 117 102 95 119 115 114 8 1400 6 120 121 103 96 122 118 115 9 1600 -7 117 120 101 95 122 117 115 10 1800 I ' - 1 119 123 101 96 123 119 116 11 2000 -8 118 124 100 96 124 120 118 | </div>

Claims

<div class="application article clearfix printTableText" id="claims"> - 16 - Table 5 mple Hbcontent Content at Content at Content at Content at Content at Content at Content at Content at [mg/dl] 405 nm 480 nm 505 nm 546 nm 570 nm 600 nm 660 nm 700 nm [U/l] [U/l] [U/l] [U/l] [U/l] [U/l] [U/l] [U/l] 1 0 88.0 93.4 93.8 92.6 90.8 92.1 93.0 91.0 2 200 83.5 91.1 99.4 97.4 90.3 94.3 91.9 93.1 3 400 68.5 95.0 97.5 90.6 92.5 95.6 • 87.1 99.3 4 600 58.0 97.0 98.7 94.4 90.2 97.6 103.6 99.4 5 800 45.0 100.2 99.1 91.3 85.6 94.2 100.7 103.0 6 1000 38.6 101.8 101.5 91.4 92.3 105.3 100.9 99.4 7 1200 38.1 101.4 101.1 90.3 87.8 104.7 98.0 100.8 8 1400 18.0 101.4 104.8 92.0 91.6 104.9 109.3 104.6 q 1600 22.6 104.0 104.4 94.9 90.3 103.0 99.4 104.7 10 1800 20.2 102.9 102.2 91.9 94.3 102.8 106.8 94.0 11 2000 j 18.7 103.4 102.7 91.8 89.2 100.3 106.2 102.0 I ample Hb content! Recovery Recovery Recovery Recovery Recovery Recovery Recovery Recovery [mg/dl] I 405 nm 480 nm 505 nm 546 nm 570 nm 600 nm 660 nm 700 nm m [%] P/o] [%] [%] [%] [%] [%] 1 o 100 100 100 100 100 100 100 100 2 200 95 98 106 105 99 102 99 102 3 400 78 102 104 98 102 104 94 109 4 600 66 104 105 102 99 106 111 109 5 800 51 107 106 99 94 102 108 113 6 1000 44 109 108 99 102 114 109 109 7 1200 43 109 108 98 97 114 105 111 8 1400 20 109 112 99 101 114 118 115 9 1600 26 111 111 102 99 112 107 115 10 1800 23 110 109 99 104 112 115 103 11 2000 | 21 111 110 99 98 109 114 112 | 04 CM 4>-CO 33 149 - 17 - 8U1" 33 1 49 1 1. Method for the determination of an analyte in a sample containing free haemoglobin by optical toichromatic measurement at a main and secondary wavelength, wherein a secondary wavelength above 475 nm is used in which absorption bands of haemoglobin are located. 2. Method as claimed in claim 1, wherein a secondary wavelength in the range of 546 ± 10 nm is used. 3. Method as claimed in claim 1, wherein a secondary wavelength in the range of 570 ± 10 nm is used. 4. Method as claimed in one of the claims 1-3, wherein a test is carried out which is based on a measurement of the increase or decrease of the concentration of NADH or NADPH in the sample. 5. Method as claimed in claim 4, wherein a main wavelength in the range of 340 ± 10 nm is used. IN I LLLfcl I UAL PROPERTY OFFICE OF N.Z. 18 FEB 1999 ..Received 33149 - 18 - 6. Method as claimed in one of the claims 1-5, wherein the content of an analyte is determined selected from the group comprising ammonia, creatine kinase and isoenzymes thereof and lactate, dehydrogenase and isoenzymes thereof. 7. Method as claimed in claim 6, wherein ammonia is determined. 8. Method as claimed in claim 6, wherein creatine kinase or/and the creatine kinase isoenzyme CK-MB is determined. 9. Method as claimed in claim 6, wherein lactate dehydrogenase or/and the lactate dehydrogenase isoenzyme HBDH is determined. 10. Method as claimed in one of the claims

1-9, wherein a sample is determined which contains a blood substitute. 11. Method as claimed in one of the claims l-l0, wherein the determination is carried out on a serum or plasma sample. 33149 1 12. Method as claimed in one of che claims l-ll, wherein the determination is carried out in an automated analyzer 13. Method for the determination of an analyte in a sample as defined in claim 1 substantially as herein described with reference to any example thereof. By fcifcVthalr autbortofd Agents A.J. PAftK & SON. end of claims INTELLECTUAL PROPERTY OFFICE OF N.Z. 18 FEB 1999 RECEIVED </div>