METHOD FOR DETERMINING THE FIBRINOGEN CONCENTRATION IN A BIOLOGICAL SAMPLE

Abstract

The present invention relates to clinical decision support systems. In detail, the present invention relates to a method for determining the initial fibrinogen concentration in a biological sample, to the use of fibrinogen added to a biological sample in at least one predetermined concentration, to a device for determining the initial fibrinogen concentration in a biological sample, to a computer program comprising program code means for causing a computer to carry out at least several steps of the method according to the invention, to a computer readable non-transitory storage medium containing instructions for carrying out at least some steps of the method according to the invention, and to a kit for determining the initial fibrinogen concentration in a biological sample.

Claims

1. Method for determining the initial fibrinogen concentration in a biological sample comprising the following steps: providing a biological sample; adding fibrinogen to said biological sample in at least one predetermined concentration; starting a clotting process in said biological sample; determining the attenuance of the biological sample as a function of time to obtain an attenuance curve; extracting feature values indicative of the fibrinogen concentration in said biological sample from said attenuance curve and recording them as a function of fibrinogen added in step; calculating a fitted function through said recorded feature values of step; determining the initial fibrinogen concentration in said biological sample from said fitted function of step.

2. The method of claim 1, characterized in that said step is realized by the following steps: extrapolating said fitted function of step to the value of added fibrinogen where said feature value is zero to obtain an extrapolated value; and/or extrapolating said fitted function of step to the value of added fibrinogen to a value that coincides with a hypothetical sample with a fibrinogen level of zero to obtain an extrapolated value; determining the initial fibrinogen concentration in said biological sample by multiplying said extrapolated value by −1.

3. The method of claim 1, characterized in that said feature values indicative of the fibrinogen concentration in said biological sample are selected from: difference in attenuance of said biological sample between a first point in time and a second point in time (Δ attenuance), preferably at the starting point of the clotting process as the first point in time, and at the end of the clotting process as the second point in time (Δ attenuance); maximum attenuance rate (time derivative of attenuance curve); features which correspond with time, including: lag time, time to (a certain degree of) maximum attenuance.

4. Method of claim 1, characterized in that said fitted function of step is selected from the group consisting of: a linear function; a non-linear function, including: exponential function, power function, reciprocal function.

5. Method of claim 1, characterized in that in step said clotting process is started by adding a clotting trigger to said biological sample, preferably selected from the group consisting of: active coagulation factors, including: thrombin (F2a), tissue factor (TF), active FX (FXa), active proconvertin (FVIIa); snake venom thrombin-like enzymes, including: batroxobin and reptilase; kaolinite, micronized silica, ellagic acid, wherein preferably said clotting trigger is added to said biological sample in a concentration sufficiently high that the shape of the attenuance curve is essentially determined by the fibrinogen concentration in the biological sample.

6. Method of claim 1, characterized in that in step said biological sample is provided in at least two aliquots allowing the execution of steps in said at least two aliquots, preferably in parallel, resulting in the obtainment of at least two attenuance curves.

7. Use of fibrinogen added to a biological sample in at least one predetermined concentration for determining the initial fibrinogen concentration in said biological sample.

8. A device for determining the initial fibrinogen concentration in a biological sample comprising: a container capable of receiving a biological sample and an addition of fibrinogen in at least one predetermined concentration; a measuring unit configured for measuring the attenuance of said biological sample; a calculating unit configured for: determining the attenuance of the biological sample as a function of time to obtain an attenuance curve; extracting feature values indicative of the fibrinogen concentration in said biological sample from said attenuance curve and recording them as a function of fibrinogen to said biological sample; calculating a fitted function through said recorded feature values; determining the initial fibrinogen concentration in said biological sample from said fitted function.

9. The device of claim 8, characterized in that said calculating unit is further configured for: extrapolating said fitted function to the value of added fibrinogen where said feature value is zero to obtain an extrapolated value and/or extrapolating said fitted function to the value of added fibrinogen to a value that coincides with a hypothetical sample with a fibrinogen level of zero to obtain an extrapolated value; determining the initial fibrinogen concentration in said biological sample by multiplying said extrapolated value by −1.

10. The device of claim 8, characterized in that said container is a multi-well container capable for receiving at least two aliquots of said biological sample, preferably a microtiter plate or a cartridge with multiple fluid chambers.

11. The device of claim 8, characterized in that said measuring unit is an optical measuring system comprising at least one light source and at least one light detector.

12. The device of claim 8, characterized in that said calculating unit comprises a processor and a computer-readable storage medium, wherein said computer-readable storage medium contains instructions for execution by said processor, wherein preferably said instructions cause said processor to perform the steps of: determining the attenuance of the biological sample as a function of time to obtain an attenuance curve; extracting feature values indicative of the fibrinogen concentration in said biological sample from said attenuance curve and recording them as a function of fibrinogen to said biological sample; calculating a fitted function through said recorded feature values; determining the initial fibrinogen concentration in said biological sample from said fitted function, and preferably extrapolating said fitted function to the value of added fibrinogen where said feature value is zero to obtain an extrapolated value and/or extrapolating said fitted function to the value of added fibrinogen to a value that coincides with a hypothetical sample with a fibrinogen level of zero to obtain an extrapolated value; determining the initial fibrinogen concentration in said biological sample by multiplying said extrapolated value by −1.

13. Computer program comprising program code means for causing a computer to carry out at least the steps, and of the method of claim 1 when said computer program is carried out on the computer.

14. A computer-readable non-transitory storage medium containing instructions for execution by a processor, wherein the instructions cause the processor to carry out at least the steps, and of the method of claim 1.

15. A kit for determining the initial fibrinogen concentration in a biological sample comprising: the device and; fibrinogen, preferably in solution comprising a predetermined concentration thereof; a manual for performing the method of claim 1; optionally a clotting trigger, preferably selected from the group consisting of: active coagulation factors, including: thrombin (F2a), tissue factor (TF), active FX (FXa), active proconvertin (FVIIa); snake venom thrombin-like enzymes, including: batroxobin and reptilase; kaolinite; further optionally

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0068] In the drawings:

[0069] FIG. 1 shows a typical example of attenuance or optical density or turbidity change (on the vertical axis) measured over time (horizontal axis) in a clotting plasma sample initiated by thrombin. After a short lag time in which fibrinogen is converted to fibrin, activated fibrin monomers start to polymerize and form thick fibrin fibers that cause light to scatter which is detectable by a decrease in transmitted light or increase of turbidity or attenuance;

[0070] FIG. 2 shows the Δ attenuance in the sample and in sample with the addition of 0.5, 1, 1.5, 2 g/L of fibrinogen (open circles). A linear function can be used to fit the measurement points. An extrapolation of the linear fit to the intersection with the horizontal axis, which corresponds with the Δ attenuance in case no fibrinogen would be present in the sample, indicates that the sample's initial fibrinogen concentration is 3.14159 g/L;

[0071] FIG. 3 shows the Δ attenuance observed in fibrinogen deficient plasma added with 1, 2, 3 and 4 g/L of fibrinogen and triggered with 0.02 (o), 0.06 (+) and 1 (x) NIH U/mL. The fitted parameters of the linear function were used to calculate the fibrinogen concentration in the sample using the herein described method and resulted in 0.12 (95%—confidence interval: −0.013-0.24), 0.16 (95%—CI: 0.006-0.29) and 0.033 (95%—CI: —0.074-0.13) g/L fibrinogen, respectively;

[0072] FIG. 4 shows the observed changes of lag time in seconds of the attenuance curve using a simulated fibrinogen addition experiment ranging from −3.1 (corresponding to fibrinogen extraction) to 0.5 g/L of a sample containing 3.14159 g/L fibrinogen. A reciprocal fit of the form lag time (fg.sub.add)=85.7234/(fg.sub.add−3.1416) was found to fit the observations closely. The vertical asymptote is found at −3.1416, hence the determined concentration of fibrinogen in the sample is 3.1416 g/L which is in good agreement with the actual fibrinogen level;

[0073] FIG. 5 schematically shows a respective flow diagram of an exemplary embodiment of the method according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0074] The clotting of the plasma can be initiated by for example tissue factor, kaolin, thrombin or another clotting agent or trigger, respectively. During this clotting, the opacity of a plasma sample changes. This will result in a decrease in light transmission which can be measured by means of the attenuance, optical density or the turbidity of the sample over time (Carr and Gabriel, Macromolecules (1980) 13(6): 1473-1477). The amount of fibrinogen in a sample has a high influence on the shape of the attenuance, optical density or turbidity curve, and the clotting assay can be developed in such a way that at sufficiently high concentration of thrombin or another enzyme with a similar activity towards fibrinogen differences between the attenuance or optical density curves of plasma samples are mainly due to their difference in the fibrinogen concentration. See Error! Reference source not found. for a typical attenuance curve example of a plasma sample in which clotting is initiated with thrombin. Features extracted from an attenuance curve are therefore indicative of the fibrinogen level in the sample and can be used to construct a calibration curve using addition of known fibrinogen levels to the sample.

[0075] In the method according to the invention, the calibration curve based on an external control plasma with a known fibrinogen concentration is replaced by a calibration in the sample of interest itself by the addition of at least one, but preferably several, known fibrinogen concentration(s), preferably to the initial sample. The features of interest from the attenuance curve are extracted from the multiple measurements.

[0076] FIG. 2 shows the theoretical difference in attenuance of the sample (as calculated in a model) between the start and end of the measurement, further referred to as A attenuance, as a function of the concentration of added fibrinogen (here in grams fibrinogen added per liter plasma sample). The Δ attenuance is a preferred feature from the attenuance curve exemplarily used in this invention. It can be seen in FIG. 2 that this feature varies linearly with added fibrinogen. If no fibrinogen would be present in the sample before adding any fibrinogen, there would be no attenuance change at zero added fibrinogen because no fibrin could be formed. The non-zero value of Δ attenuance of the original sample, i.e. at zero added fibrinogen, indicates the initial concentration of fibrinogen in the sample, i.e. the value that is to be determined by the method according to the invention.

[0077] The measurement of a clotting feature, e.g. Δ attenuance, at zero and one or more non-zero values for added fibrinogen is therefore followed by a step that involves the calculation of a fit, sometimes referred to as regression, through the multiple observed feature values. Extrapolation of the fitted curve then yields the original fibrinogen concentration in the plasma sample. For the Δ attenuance feature this works as follows: [0078] estimate a linear fit (Δ attenuance=α*fg.sub.add+β, with fg.sub.add the added fibrinogen and α and β fitting parameters, respectively slope and offset of the linear fit); [0079] extrapolate the fitted function to the value of added fibrinogen where the Δ attenuance is zero. This will be a negative value (or zero); [0080] the fibrinogen level in the sample is calculated as minus the extrapolated value. For the linear relationship with added fibrinogen that is followed by Δ attenuance this equals the offset of the line divided by the slope (fg.sub.sample=−β/α).

[0081] The above method with Δ attenuance as the observed feature was applied to a sample of fibrinogen deficient plasma, i.e. the fibrinogen level in such a sample is closed to zero. In this example the sample was found to have a residual fibrinogen antigen concentration of 0.0 5 g/L (detected with ELISA). Next, clotting in the fibrinogen deficient plasma was initiated with multiple thrombin concentrations (0.02, 0.06 and 1 NIH U/mL) in combination with multiple fibrinogen additions (1, 2, 3 and 4 g/L). The turbidity or attenuance for every combination was measured in four replicates for each of the (12) experimental combinations. The attenuance curves were averaged and the Δ attenuance was calculated for each experimental condition, see FIG. 3.

[0082] Next step was to fit a linear function to each of the thrombin concentrations as discussed previously (see FIG. 2). Next, the fibrinogen concentration of the original sample was inferred by using the before mentioned equation. This resulted in a fibrinogen level of 0.12 (95%-confidence interval; CI: —0.013-0.24), 0.16 (95%—CI: 0.006-0.29) and 0.033 (95%—CI: —0.074-0.13) g/L for the 0.02, 0.06 and 1 NIH U/mL of thrombin, respectively, which corresponds well with the expected fibrinogen concentration. The best results, i.e. closest to the actual fibrinogen level as determined by ELISA as well as smallest confidence interval, were obtained with 1 NIH U/mL of thrombin which is likely due to minimal impact of the feedback in the coagulation system on the clotting process.

[0083] Alternatively a similar approach can be followed for other extracted features of the attenuance curve that possibly demonstrate non-linear behavior for multiple fibrinogen additions. For example, features that correspond with time, e.g. lag time to (95% of) maximum attenuance, time to maximum rate (i.e. the time at which the maximum of the time derivative of the curve occur), can be fitted with e.g. an exponential, power or reciprocal function with added fibrinogen as the variable. Such features go to infinity as the true concentration of fibrinogen in the sample goes to zero as can be seen in a simulated experiment with the computer model, see FIG. 4. The horizontal (x−) coordinate of the (vertical) asymptote of the fitted curve relates to the negative value of the fibrinogen level in the sample. In case a reciprocal function of the form α/(fg.sub.add)−β) is fitted, which is preferred in case of the features related to points in time, with fg.sub.add the level of added fibrinogen and α, β unknown parameters that are fitted with an algorithm, −β equals the fibrinogen level of the sample as the vertical asymptote is found at β. Another feature, the maximum rate of the attenuance curve is preferably fitted with a function of the form α*(fg.sub.add−β).sup.2, with fg.sub.add the level of added fibrinogen and α, β unknown parameters that are fitted with an algorithm. In that case the minimum of the quadratic function can be found at β, hence −β equals the fibrinogen level of the sample as well.

[0084] Alternatively, features extracted from the average mass/length curve can be used to infer the fibrinogen concentration. For example, the maximum rates observed in the samples with added fibrinogen indicate a linear relationship between added fibrinogen and maximum rate (i.e. the maximum of the time derivative of the curve). This relation can similarly be used to infer the fibrinogen concentration of the sample. Features extracted from the average mass/length ratio pertaining to the dynamics of the curve, i.e. lag time, time to maximum rate (i.e. the maximum of the time derivative of the curve), time to (95% of) the maximally achieved mass/length ratio, can be approximated best by a reciprocal function or alternatively by a power or exponential function. The fibrinogen level of the sample can then be inferred by finding the (vertical) asymptote, as discussed previously.

[0085] FIG. 5 schematically shows a respective flow diagram of an embodiment of the method according to the invention. In step (S1) a biological sample, such as a blood plasma sample of an individual suspected of having an altered fibrinogen concentration, is provided. In step (S2) the clotting process is started in said biological sample e.g. by the addition of a clotting trigger, such as thrombin (F2a) or snake venom thrombin-like-enzymes, respectively. In step (S3) fibrinogen is added in at least one predetermined concentration to said biological sample. It is preferred that multiple amounts of fibrinogen are added to aliquots of the sample in parallel experiments, for example divided over a cartridge with multiple detection chambers. In step (S4) the attenuance(-s) of the biological sample(s) containing the added fibrinogen concentration(s) is/are determined as a function of time and a/several attenuance curve(s) is/are obtained.

[0086] In step (S5) feature values indicative of the fibrinogen concentration in said biological sample, such as the Δ attenuance, are extracted from said attenuance curve(s) and recorded as a function of fibrinogen that was added in step (S3). In step (S6) a fitted function is calculated through said recorded feature values of step (S5). In step (S7.1) said fitted function of step (S6) is extrapolated to the value of added fibrinogen where said feature value is zero or alternatively in case of features related to timing of the clotting process the value of the vertical asymptote or alternatively in case of maximum rate of the attenuance curve the value in which the quadratic fitting function has its minimum value, to obtain an extrapolated value. In step (7.2) the initial fibrinogen concentration in said biological sample is determined by multiplying said extrapolated value by −1.

[0087] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplarily and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims.

[0088] In the claims, the word “comprising” does not exclude other elements or steps, and the undefined article “a” or “an” does not exclude a plurality. A single element or other unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.