Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
1997-11-03
2001-07-31
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C356S320000, C436S175000
Reexamination Certificate
active
06268167
ABSTRACT:
The present invention concerns a method for the analysis of a medical sample while avoiding measurement errors due to haemolysis.
The most common test material for biochemical analyses is blood serum or plasma. It is known that errors can occur in the determination of various analytes from blood serum or blood plasma which influence the result of the measurement and which are caused by the properties of the sample. Measurement errors in this sense are caused in particular by hemolytic sample material. Hemolysis in blood samples can substantially falsify the analytical measurement results.
If hemolysis occurs in a blood sample then some of the red blood corpuscles have been destroyed and the constituents released in this process contaminate the sample material. As a consequence of this the analytical serum or plasma values are rendered inaccurate by the constituents of the blood corpuscles such as e.g. haemoglobin.
In order to eliminate this disadvantage U.S. Pat. No. 4,263,512 proposes the determination of the interfering chromogen in a blood sample together with the analyte and recommends correction of the measurement error according to the extent of haemolysis (correlates with the chromogen concentration). However, this conventional correction method only takes into account the red blood pigment released by the erythrocytes in order to determine the measurement error in haemolytic sample material. EP-0 268 025 B1 points out that there is a quantitative relationship between the extent of interference by haemolysis, the analyte concentration and the measurement error caused by the interference. This relationship can be expressed with the aid of multiple regression. The correction factors derived from this fit then enable the analytical result to be corrected on the basis of a hemolysis interference determined independently e.g. by measurement of the Hb value (while ignoring other possible dependencies on analyte concentration).
The clinical chemical analyzer “Synchron CX 5” from the Beckman Company utilizes a set of 2 to 5 wavelengths to measure reaction time courses and to compensate for interfering background. Suitable selection of additional, non-reaction-relevant wavelengths enables an automatic correction for endogeneous spectral interferences. At 16 second intervals an eight flash photometric measurement is carried out, i.e. in the case of a maximum of 5 wavelengths 8×5=40 mesurement data are generated. The following polychromatic equation is then used for the signal correction:
Absorbance difference=difference(
A−B−C−D−E+k
)
in which
A=change in the absorbance of the bichromatically measured analyte reaction
B-E=weighted bichromatic correction wavelengths and
k=constant which enables the spectral influence of sera in the absence of lipemia, haemolysis or icterus to be taken into account.
Depending on the automated analyzer, the polychromatic correction can either be carried out categorically (Beckman analyzer) when it is part of a test application or only when a test-specific limit has been exceeded above which the interference error has a marked influence on sample recovery.
However, the object of the present invention is to provide an analytical method which enables measurement errors caused by contaminating components in a blood serum sample or in a blood plasma sample of haemolytic blood to be determined with improved accuracy and considerably less laboriously than conventional correction methods.
This object is achieved by a method for analysing a medical sample while avoiding measurement errors due to hemolysis in which, before the actual photometric determination of a component present in the sample, the sample is subjected to a pre-reaction in which the extent of hemolysis in the sample is determined and the measured value of the component to be determined obtained subsequently is corrected by a value which has been determined by correlating the extent of hemolysis with the measurement error contribution of interfering components.
A distinguishing feature of the method according to the invention compared to the state of the art is the advantage that an independent determination of the degree of hemolysis, e.g. by determining the haemoglobin value in a sample, is not necessary for the correction of the measured value of a haemolytic sample. Moreover a relationship was found between the degree of hemolysis of a sample and the pre-reaction determined in hemolytic sera or plasma. In this case the pre-reaction occurs in the presence of sample and a reagent but before adding the start reagent of the actual measurement determination.
It is possible with the aid of the biometric model described in the present invention to directly estimate the degree of hemolysis of a sample from the pre-reaction which it causes and to carry out a corresponding correction of the analytical result. A further contribution to the error which must be taken into consideration for the analysis is the fact that an incorrect result for hemolytic samples can also be obtained when determining particular substances when this substance is also present in red blood corpuscles and as a result is also additionally present in the sample to be analysed after hemolysis. This contribution to the measurement error is also taken into account by the method according to the invention since, in addition to the degree of hemolysis, the content of analyte in the sample also enters into the pre-reaction.
The method according to the invention enables correction of measurement errors due to hemolysis and is particularly efficient at correcting in the reference range (31 U/l in women, 37 U/l in men) (<10% increase in recovery (see FIG.
1
)).
In addition, beyond the reference range, interferences of up to an order of magnitude of 80% obtained with methods according to the state of the art could be reduced to less than 30% using the appropriate correction.
In the method according to the invention the pre-reaction alone takes into account the individual properties of the sample so that, on the basis of the previous experimental data, it is unnecessary to carry out further specific correction processes for the sample material. In particular additional measurements are not required. This is an important advantage compared to the previously described correction methods for hemolytic sera or plasma.
In a preferred embodiment of the invention the pre-reaction is also determined photometrically. In this case it is particularly preferred that the photometric determination is carried out bichromatically and in particular at {fraction (340/405+L )} nm and that the actual measured value is derived from the difference between the results of the two wavelengths.
The pre-reaction is preferably started by addition of a reagent which contains NADH and LDH (lactate dehydrogenase). In a particularly preferred embodiment the reagent additionally contains MDH (malate dehydrogenase).
In a further preferred embodiment of the present invention the reliability of the relationship between the degree of hemolysis determined by the pre-reaction and the contribution to the measurement error caused by the interfering components such as hemoglobin in particular or by the component to be determined which is, however, present in the sample as a result of haemolysis is ensured on a broad basis for a large group of test persons with a heterogeneous state of health, age and sex.
In a particularly preferred method the relationship between the pre-reaction and the main reaction with regard to the component to be determined is defined with the aid of the formula
rate
substance/sample
=rate
total
−rate
pre-reaction
−rate
substance/erythrocytes
in which substance denotes the component to be determined in the sample.
One possibility for eliminating interference can be ascertained with the aid of a mathematical relationship between the pre-reaction and the main reaction. A schematic reaction time course is shown in FIG.
2
and the mathematical relationship for the
Berding Christoph
Kleider Wilhelm
Weber Friederike
Wild Thomas
Fulbright & Jaworski LLP
Gitomer Ralph
Roche Diagnostics GmbH
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