The present invention relates to a method of determining the concentration of heparin in fluid samples, and to a device for performing the method.
Heparin is a heteropolysaccharide of the acid mucopolysaccharide type which has anticoagulant activity resulting from its ability to catalyze the reaction between antithrombin III and thrombin. On the basis of this activity, heparin is widely used as an anticoagulant in cardiovascular surgery, for example, during operations with extracorporeal circulation, and in other diagnostic and therapeutic applications.
When heparin is used as an anticoagulant, it may be very useful to have available a method which permits rapid, reproducible and accurate measurement of the concentration of heparin present in the sample of interest, and a device for performing this measurement.
In order to perform this measurement, it is known to use dyes of the cationic thiazines series. As a result of the reaction of these dyes with heparin, it is possible to observe a change in the absorption (or transmission) spectrum of the dye, that is, a reduction in the absorption due to the free dye in solution and the appearance of an absorption band of the heparin-dye complex formed. The absorption spectrum of cationic thiazines in dilute aqueous solution is in fact characterized by a main absorption band due to the contribution of the monomer and the dimer of the dye in solution. When heparin is added to this dilute solution, a second band (called the xcexc band) which corresponds to the heparin-dye complex formed appears in the absorption spectrum of the solution. The addition of heparin to the dye solution not only causes the appearance of the xcexc band, but also causes a reduction in the absorption of the main band. The reduction in the absorption of the free dye in solution and the increase in the absorption of the dye-heparin complex constitute the two components of a phenomenon known by the term xe2x80x9cmetachromasiaxe2x80x9d, the magnitudes of which, in suitable reaction conditions, can be correlated with the concentration of heparin present in the sample.
On the basis of this principle, assays have been developed for measuring the concentration of heparin in samples of interest. U.S. Pat. No. 4,911,549 (Karkar) describes a method of determining the heparin concentration in blood plasma by metachromatic reaction with the dye Azure A which belongs to the cationic thiazines series. This assay is based on the measurement of two distinct transmittance signals for two distinct wavelengths, one of which is substantially insensitive to the heparin dilution.
It has surprisingly been found that the measurement of the entire absorption spectrum of the dye in a metachromatic heparin assay, rather than measurement of distinct absorption signals with predetermined separate wavelengths, achieves results which are more accurate and reproducible and, above all, are independent of the reaction medium. In the case of assays performed on samples of biological fluids such as blood plasma containing proteins and other chemical species which could potentially interfere with accurate measurement of the absorption at these predetermined wavelengths, this latter characteristic is of substantial importance because it makes possible a device which can perform the assay automatically and which does not require repeated calibrations.
Accordingly, the invention provides a method for determining the concentration of heparin in a fluid sample comprising: (a) providing a fluid sample containing heparin; (b) adding to the fluid sample a solution of a dye to form a mixture of sample and dye, wherein the dye interacts with the heparin in the sample so that the absorption spectrum of the mixture of sample and dye in the visible range varies as a result of the interaction in a manner quantitatively dependent on the heparin concentration; (c) determining the absorption spectrum in the visible range of the mixture of sample and dye; and (d) calculating a spectral parameter representative of both the reduction in the absorption of the free dye in solution and the increase in the absorption of the dye-heparin complex, the value of the spectral parameter being indicative of the concentration of heparin present in the fluid sample, in order to determine the concentration of heparin present in the fluid sample. The relationship between the value of the spectral parameter and the concentration of the heparin present in the fluid sample has been previously determined by determining the absorption spectra, in the visible range, of a composition comprising the fluid and the dye, in the absence of heparin and in the presence of a plurality of concentrations of heparin, and calculating the relationship between the value of the spectral parameter and the concentration of heparin. A device for performing this method is also provided.
Additional features and advantages of the invention are set forth in the description which follows and in part will be apparent from the description. The objectives and other advantages of the invention will be realized and attained by the method of determining the concentration of heparin in a fluid sample and the device for performing this method as particularly pointed out in the written description and claims.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.