Chemistry: analytical and immunological testing – Hemoglobin – myoglobin – or occult blood
Reexamination Certificate
1998-11-20
2001-03-27
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Hemoglobin, myoglobin, or occult blood
C436S086000, C436S088000, C436S084000, C436S071000, C356S039000
Reexamination Certificate
active
06207459
ABSTRACT:
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 measurement and the measured value for the analyte concentration is mathematically corrected. In particular this method is suitable for the determination of the parameters total protein, iron and albumin in a medical sample e.g. in a serum or plasma sample.
It is generally known that haemolysis interferes in some cases to a considerable extent with the determination of numerous analytes. In order to nevertheless obtain measurement values that are not falsified various methods were published in the past for the reduction of haemolysis interference.
As mentioned in the patent EP-0 268 025 B1 a graphical relationship was established for some analytes between the degree of haemolysis and the resulting measurement error. Correction factors could be derived from this which were used to mathematically correct the analytical result obtained on the basis of a separate determination of the degree of haemolysis.
Jay and Provasek also describe that unfalsified values can be obtained in haemolytic samples by determining the degree of haemolysis 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 haemolysis was determined by a separate measurement of the Hb content in the sample.
In the patent document U.S. Pat. No. 4,263,512 it is recommended that the degree of turbidity (X), haemolysis (Y) and icterus (Z) are determined in addition to the analyte and that the measured analyte value (S) is corrected with the aid of the formula S′=S−&agr;.X-&bgr;.Y&ggr;.Z. In this case S′ is the corrected analyte value and &agr;, &bgr; and &ggr; are correction factors which were obtained by measuring the influence of turbidity, haemolysis and icterus 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 consideration the respective proportion of the other interfering substances.
One method of correcting haemolysis interferences without a separate determination of the degree of haemolysis is shown by DE 44 27 492 A1. Here a mathematical relation was found between the content of interfering substance released by haemolysis from the erythrocytes and a pre-reaction which occurs before the main reaction. The analytical result obtained in the main reaction (rate
total
) can be corrected with the aid of the degree of haemolysis determined in this way during the pre-reaction utilizing the relation found between the degree of haemolysis and interference contribution according to the formula rate
substance/sample
=rate
total
−rate
pre-reaction
−rate
substance/erythrocyte
, in which substance means the component to be determined in the sample.
Frequently an interference by haemoglobin can also be eliminated by measuring the sample blank value. 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 not to the determination of albumin by means of the bromocresol-green and bromocresol-purple method. Furthermore it is known that interferences by haemoglobin always occur in the determination of iron if Hb has not previously been removed from the sample 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 Hb interference by the sole measurement of the sample blank value.
However, all the methods for eliminating interference described above have the disadvantage that they involve a considerable amount of work (sample preparation by dialysis or the separate determination of the degree of haemolysis e.g. by determining the Hb content) and/or complicated mathematical correction algorithms.
In addition the described methods all relate to the elimination of erroneous measurements caused by haemolysis. The development of blood substitutes based on haemoglobin has made the issue of removing interferences by native or synthetic haemoglobin or haemoglobin-like compounds even more critical than before. Such interference then also on the one hand occur in non-haemolytic sample material and on the other hand also to a much greater extent than in the case of native haemolysis since in blood substitute treatment the haemoglobin content in blood serum or plasma can be more than 1000 mg/dl.
The object of the present invention was to provide a method for the elimination of interferences which are caused by native haemoglobin or blood substitutes based on synthetic Hb or Hb-like compounds and which cannot be eliminated by the simple measurement of the sample blank value. In addition this method should be associated with a significantly reduced work load compared to conventional methods and guarantee an elimination of interference up to at least 1000 mg/dl Hb.
The object of the invention was achieved in that surprisingly a relation was found between the level of the sample blank value and the degree of the falsification of the measured results caused by free haemoglobin. As a result it is possible with the aid of a simple mathematical correction formula to exactly determine the correct value for the analyte concentration even when the Hb content is high. In comparison to the state of the art the method according to the invention is advantageously characterized in that neither a separate determination of the Hb content nor a determination of the degree of a pre-reaction are necessary in order to correct the measured value of a medical sample containing Hb.
Hence a subject matter of the present invention is a method for the determination of an analyte in a sample containing free haemoglobin by optical measurement wherein the measured value for the analyte concentration is corrected by the steps:
(a) measuring the blank value of the sample to be analysed,
(b) measuring the blank value of a haemoglobin-free reference sample,
(c) measuring the uncorrected value for the analyte concentration and
(d) correcting the value obtained in step (c) by correlation with the values obtained in step (a) and (b) in order to obtain the corrected value for the analyte concentration.
The corrected value for the analyte concentration in step (d) of the method according to the invention is preferably determined according to the following relationship:
C′
sample
=C
sample
−F.E
1
sample
+F.E
1
reference
in which
C′
sample
is the corrected value for the analyte concentration,
C
sample
is the uncorrected measured value for the analyte concentration in the sample,
F is a test-specific correction factor,
E1
sample
is the measured blank value in the sample and
E1
reference
is the measured blank value in the reference sample.
The correction method according to the invention is suitable for methods in which the analyte is determined by optical measurement in particular at wavelengths at which an interference by free haemoglobin 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 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 derivatized, polymerized, modified or cross-linked derivatives of haemoglobins, in particular of human haemoglobin or bovine haemoglobin as well as recombinantly produced haemoglobin.
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
Schellong Lieselotte
Weisheit Ralph
Arent Fox Kintner & Plotkin & Kahn, PLLC
Roche Diagnostics GmbH
Tran Huan
Warden Jill
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