阅读说明：本技术 用于确定生物样本中纤维蛋白原浓度的方法 (Method for determining fibrinogen concentration in a biological sample ) 是由 H·J·范奥义任 B·J·巴克 R·范德汉姆 于 2015-12-18 设计创作，主要内容包括：本发明涉及临床决策支持系统。详细地讲,本发明涉及用于确定生物样本中初始纤维蛋白原浓度的方法、以至少一个预定浓度添加到生物样本的纤维蛋白原的用途、用于确定生物样本中初始纤维蛋白原浓度的设备、包括用于令计算机执行根据本发明的方法的至少几个步骤的程序代码单元的计算机程序、包含用于执行根据本发明的方法的至少一些步骤的指令的计算机可读非瞬态存储介质、以及用于确定生物样本中初始纤维蛋白原浓度的套件。(The present invention relates to a clinical decision support system. In detail, the present invention relates to a method for determining an 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 an initial fibrinogen concentration in a biological sample, to a computer program comprising program code means for causing a computer to perform at least several steps of a method according to the present invention, to a computer readable non-transitory storage medium comprising instructions for performing at least some steps of a method according to the present invention, and to a kit for determining an initial fibrinogen concentration in a biological sample.)

1. A method for determining an initial fibrinogen concentration in a biological sample, comprising the steps of:

(1) providing a biological sample;

(2) adding fibrinogen to the biological sample at least one predetermined concentration;

(3) initiating a clotting process in the biological sample;

(4) determining the attenuation of the biological sample as a function of time to obtain an attenuation curve, the biological sample comprising fibrinogen added in step (2);

(5) extracting a characteristic value from the attenuation curve indicative of the fibrinogen concentration in the biological sample and recording the characteristic value as a function of the fibrinogen added in step (2);

(6) calculating a fitting function from the feature values recorded in step (5);

(7) determining the initial fibrinogen concentration in the biological sample according to the fitted function of step (6).

2. The method according to claim 1, characterized in that said step (7) is carried out by the steps of:

(7.1) extrapolating the fitting function of step (6) to the value of added fibrinogen where the characteristic value is zero to obtain an extrapolated value;

and/or

Extrapolating the fitting function of step (6) to the value of added fibrinogen for values consistent with a hypothetical sample having a fibrinogen level of zero to obtain an extrapolated value;

(7.2) determining the initial fibrinogen concentration in the biological sample by multiplying the extrapolated value by-1.

3. The method according to claim 1 or 2, characterized in that the characteristic value indicative of the fibrinogen concentration in the biological sample is selected from:

a difference in attenuation of the biological sample between a first time point and a second time point (delta attenuation), preferably a difference in attenuation of the biological sample between a start of a coagulation process as the first time point and an end of a coagulation process as the second time point (delta attenuation);

maximum decay rate (time derivative of decay curve);

features corresponding to time, including: lag time, time to (a certain degree of) maximum attenuation.

4. The method according to any of the preceding claims, wherein the fitting function of step (6) is selected from the group comprising:

a linear function;

a non-linear function comprising: exponential function, power function, reciprocal function.

5. The method according to any one of the preceding claims, wherein in step (3) the coagulation process is started by adding a coagulation trigger to the biological sample, the coagulation trigger preferably being selected from the group comprising:

an active coagulation factor comprising: thrombin (F2a), Tissue Factor (TF), active fx (fxa), active prothrombin (FVIIa);

a snake venom thrombin-like enzyme comprising: batroxobin and reptilase;

kaolinite, micronized silicon dioxide, ellagic acid,

wherein preferably said coagulation trigger is added to said biological sample in a sufficiently high concentration such that the shape of said decay curve is substantially determined by said fibrinogen concentration in said biological sample.

6. The method according to any one of the preceding claims, wherein in step (1) the biological sample is provided in at least two aliquots, allowing to perform steps (2) - (4) in said at least two aliquots, preferably in parallel, so as to obtain at least two attenuation curves.

7. Use of fibrinogen added to a biological sample in at least one predetermined concentration to determine an initial fibrinogen concentration in the biological sample.

8. An apparatus for determining an initial fibrinogen concentration in a biological sample, comprising:

(1) a container capable of receiving a biological sample and at least one addition of fibrinogen at a predetermined concentration;

(2) a measurement unit configured to measure an attenuation of the biological sample containing the added fibrinogen;

(3) a computing unit configured to:

determining the attenuation of the biological sample as a function of time to obtain an attenuation curve;

extracting a characteristic value from the attenuation curve indicative of the fibrinogen concentration in the biological sample and recording the characteristic value as a function of fibrinogen added to the biological sample;

calculating a fitting function from the recorded characteristic values;

determining the initial fibrinogen concentration in the biological sample according to the fit function.

9. The device of claim 8, wherein the computing unit is further configured to:

extrapolating the fitting function to the value of added fibrinogen where the characteristic value is zero to obtain an extrapolated value; and/or

Extrapolating the fitting function to the added fibrinogen value for values that correspond to hypothetical samples having a fibrinogen level of zero to obtain extrapolated values;

determining the initial fibrinogen concentration in the biological sample by multiplying the extrapolated value by-1.

10. The apparatus of claim 8 or 9, wherein the container is a multi-well container capable of receiving at least two aliquots of the biological sample, preferably a microtiter plate or a kit with multiple fluid chambers.

11. The apparatus according to any of the preceding claims, characterized in that the measuring unit is an optical measuring system comprising at least one light source and at least one light detector.

12. The device according to any of the preceding claims, wherein the computing unit comprises a processor and a computer-readable storage medium, wherein the computer-readable storage medium contains instructions for execution by the processor, wherein preferably the instructions cause the processor to perform the steps of:

determining an attenuation of the biological sample as a function of time to obtain an attenuation curve;

extracting a characteristic value from the attenuation curve indicative of the fibrinogen concentration in the biological sample and recording the characteristic value as a function of fibrinogen added to the biological sample;

calculating a fitting function from the recorded characteristic values;

determining the initial fibrinogen concentration in the biological sample from the fitted function, and preferably,

extrapolating the fitting function to the value of added fibrinogen where the characteristic value is zero to obtain an extrapolated value;

and/or

Extrapolating the fitting function to the added fibrinogen value for values that correspond to hypothetical samples having a fibrinogen level of zero to obtain extrapolated values;

determining the initial fibrinogen concentration in the biological sample by multiplying the extrapolated value by-1.

13. Computer program comprising program code means for causing a computer to carry out at least the steps (4), (5), (6), (7), (7.1) and (7.2) of the method as claimed in any one of claims 1 to 6 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 perform at least steps (4), (5), (6), (7), (7.1) and (7.2) of the method according to any one of claims 1 to 6.

15. A kit for determining an initial fibrinogen concentration in a biological sample, comprising:

the apparatus of any one of claims 8-12;

fibrinogen, preferably in a solution comprising a predetermined concentration thereof;

a manual for performing the method according to any one of claims 1-6;

optionally, optionally

A coagulation trigger, preferably selected from the group comprising:

an active coagulation factor comprising: thrombin (F2a), Tissue Factor (TF), active fx (fxa), active prothrombin (FVIIa);

a snake venom thrombin-like enzyme comprising: batroxobin and reptilase;

kaolinite;

and also optionally, the step of (a) further comprising,

the computer-readable non-transitory storage medium of claim 14.

Technical Field

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

Background

Fibrinogen is an important protein involved in blood coagulation. Fibrinogen is soluble in normal blood flow; however, once the coagulation system is activated, fibrinogen is eventually converted to fibrin by thrombin. Fibrin then polymerizes into insoluble fibrin fibers, preferably forming a clot with activated platelets. Normal fibrinogen concentrations are about 2.5g/l, ranging from about 1.5 to 3g/l [ Greer et al (eds.). Wintrobe's Clinical Hematology 11 th edition, page 720 ]. However, in many cases, fibrinogen concentrations may exceed normal ranges, which may be associated with pathological conditions. For example, in patients with hereditary hypofibrinogenemia, a dangerous situation resulting from a low fibrinogen concentration due to continuous bleeding can be dealt with by adding a blood product. On the other hand, elevated fibrinogen concentrations were found to be associated with increased risk of myocardial infarction (Bom et al, aridioscler. Thromb. Vasc. biol. (1998)18(4): 621-. The fibrinogen concentration test is a valuable clinical test due to these varying fibrinogen concentrations and associated pathologies. Unfortunately, however, there is no reliable point of care (POC) fibrinogen testing and central laboratory test appointments often take too long for time critical situations (e.g., in an operating room).

A number of methods have been developed to accurately detect fibrinogen levels in plasma or blood samples (see, for example, Palarati et al Clinical Chemistry (1991)37 (5): 714-719 to obtain a summary of available techniques, or EP 2259069).

Unfortunately, all currently available methods are either very labor intensive, e.g., coagulation recovery methods, or require that a calibration curve be derived from a plasma sample with known fibrinogen levels to infer the fibrinogen level of the sample, e.g., Clauss assay or thrombin time derivation methods. The former involves a significant amount of manual time and is therefore difficult to automate, while the latter method requires the inclusion of calibration plasma in the test protocol. A method requiring at least two plasma reference samples to calibrate the measurement results is disclosed in EP 0059277. However, these methods are not suitable for incorporation into automated equipment, such as point of care (POC) handheld devices, to detect fibrinogen levels.

Disclosure of Invention

It is an object of the present invention to provide a method for determining an initial fibrin concentration in a biological sample, by means of which the disadvantages of the prior art methods can be avoided. In particular, a method for determining an initial fibrinogen concentration in a biological sample shall be provided that allows to reliably determine whether a subject biological sample is bleeding prone or at risk of thrombosis or other pathologies associated with fibrinogen levels in a body fluid (e.g. blood or plasma) of the subject or patient.

It is another object of the present invention to provide: an apparatus for determining an initial fibrinogen concentration in a biological sample; a computer program comprising program code means for causing a computer to carry out at least some of the steps of the method according to the invention; a computer-readable non-transitory storage medium containing the computer program; and a kit for determining an initial fibrinogen concentration in a biological sample.

In a first aspect of the present invention, a method for determining an initial fibrinogen concentration in a biological sample is presented, the method comprising the steps of:

(1) providing a biological sample;

(2) adding fibrinogen to the biological sample at least one predetermined concentration;

(3) initiating a clotting process in the biological sample;

(4) determining an attenuation of the biological sample as a function of time to obtain an attenuation curve;

(5) extracting from the attenuation curve characteristic values indicative of the fibrinogen concentration in the biological sample and recording them as a function of the fibrinogen added in step (2);

(6) calculating a fitting function from the feature values recorded in step (5);

(7) determining the initial fibrinogen concentration in the biological sample according to the fitted function of step (6).

In another aspect of the invention, there is provided the use of fibrinogen added to a biological sample in at least one predetermined concentration for determining an initial fibrinogen concentration in said biological sample.

In a further aspect of the invention, there is provided an apparatus for determining an initial fibrinogen concentration in a biological sample, the apparatus comprising:

(1) a container capable of receiving a biological sample and at least one addition of fibrinogen at a predetermined concentration;

(2) a measurement unit configured to measure an attenuation of the biological sample;

(3) a computing unit configured to:

determining an attenuation of the biological sample as a function of time to obtain an attenuation curve;

extracting from the attenuation curve characteristic values representing the fibrinogen concentration of the biological sample and recording them as a function of fibrinogen added to the biological sample;

calculating a fitting function from the recorded characteristic values;

determining the initial fibrinogen concentration in the biological sample according to the fitting function.

In a further aspect of the invention, a computer program is provided, comprising program code means for causing a computer to carry out at least the steps (4), (5), (6), (7) of the method according to the invention.

In a further aspect of the invention, a computer-readable non-transitory storage medium is provided, containing instructions for execution by a processor, wherein the instructions cause the processor to perform at least the steps (4), (5), (6), (7) of the method according to the invention.

In a further aspect of the invention, a kit for determining an initial fibrinogen concentration in a biological sample is provided, the kit comprising a device according to the invention, fibrinogen, preferably in dry or lyophilized form, further preferably a predetermined amount of fibrinogen, a manual for performing a method according to the invention, and optionally a coagulation trigger, and further optionally a computer-readable non-transitory storage medium according to the invention.

In a further aspect of the invention, a computer program is provided comprising program code means for causing a computer to carry out the steps of the method when said computer program is carried out on the computer, and a non-transitory computer readable recording medium having stored thereon a computer program product which, when executed by a computer processor, causes the steps of the method disclosed herein to be carried out.

Preferred embodiments of the invention are defined in the dependent claims. It shall be understood that the claimed usage, device, computer program, computer-readable non-transitory storage medium and kit have similar and/or identical preferred embodiments as the claimed method and as defined in the dependent claims. All the dependent claims relating to the method according to the invention can thus also be combined with the use, the device, the computer program, the computer-readable non-transitory storage medium and the kit according to the invention and with each other.

The inventors of the present invention propose a method which makes it possible to add at least one but preferably more amount of fibrinogen to the biological sample itself in a parallel test (e.g. dispensed on a cartridge (cartridge) with multiple detection chambers in a POC device), instead of using a calibration curve constructed from several plasmas with known fibrinogen levels or a dilution series of plasmas with known fibrinogen levels. This will result in an internal calibration curve within the sample itself, rather than an external calibration curve derived from a plasma sample with known fibrinogen levels. The method according to the invention can be used automatically, for example on POC platforms with multiple chambers and with known fibrinogen content or concentration, without the need to calibrate the plasma to determine fibrinogen levels in the sample. The method according to the invention can also be implemented on a central laboratory apparatus, which results in omitting or reducing the use of calibration plasma. Further, the internal calibration has an advantage that the internal calibration can correct physical properties of the initial sample, such as matrix composition, more appropriately than the external calibration.

The use of an internal calibration curve to determine the initial concentration of fibrinogen by adding one or more fibrinogen concentrations to a biological sample is neither known nor obvious in the prior art.

According to the present invention, a "biological sample" refers to any sample suspected of containing fibrinogen. Biological samples may be of natural or unnatural origin. Preferred samples are body fluids, especially those that are capable of undergoing blood clotting or coagulation, such as blood samples, plasma samples, whole blood samples, and the like.

According to the present invention, the "initial fibrinogen concentration" refers to the concentration of fibrinogen present in the biological sample provided in step (1), i.e. before any fibrinogen is added in step (2).

According to the invention, "predetermined concentration" means that the user has exact knowledge of the added fibrinogen concentration. "adding fibrinogen" refers to adding fibrinogen to a biological sample. "add" may also refer to adding a biological sample to a predetermined concentration of fibrinogen placed in a container, for example, in fibrinogen solution or in dry or lyophilized form.

According to the invention, the "agglomeration process" refers to a process involving the conversion of fibrinogen into fibrin. This conversion preferably follows a subsequent step, for example, the polymerization of fibrin into insoluble fibrin fibers, which form blood clots with the preferably activated platelets. The process of coagulation in whole in a blood or plasma sample is also referred to as the process of blood coagulation. The coagulation process may be initiated, for example, by adding a so-called coagulation trigger to the biological sample.

According to the present invention, "attenuation" refers to a measure of absorption plus loss of a substance, such as a biological sample, for example, due to scattering and fluorescence. The attenuation of biological samples, such as coagulated plasma or blood samples, changes over time due to the formation of a fibrin network during coagulation of the sample. Fibrin fibers are formed after the addition of a coagulation trigger to the sample. These fibrin fibers cause the incident light to scatter, resulting in fewer photons reaching the detector. Other causes of the actual absorption and attenuation of photons, such as fluorescence, are considered constant over time during coagulation, so the attenuation and thus the reduction in transmission and increase in optical density is considered to be due solely to scattering of the incident light by fibrin fibers formed in the sample. The attenuation of the material is log10 (P)₀/P) where P₀Is the power of radiation incident on the sample and P is the power transmitted by it. This amount is again-log 10(T), where T is the transmission. Attenuation is usually directly related to or related to terms in the literature such as "optical density", "haze" or "extinction". Also, the name "absorption" (symbol: A) is often used incorrectly for this quantity, but this is not appropriate for this quantity when the attenuation of the radiation is due to scattering rather than absorption. This quantity is known per se as attenuation (symbol: D), noting that in scattering or reflectionWhen negligible, the attenuation degrades to absorption. The inventors have found that scientists can use the term "turbidity" when attenuation degrades to scatter, which is generally considered to be-ln (t). In the latter case, "turbidity" can be estimated by measuring transmittance. In order to actually measure scattering due to particles in solution, there are special techniques such as small angle scattering or turbidimetry. Attenuation can be converted into "turbidity", "transmittance", "optical density", "absorbance" and other measures of light attenuation or scattering due to particles in solution. Thus, according to the present invention, the aforementioned dimensions or terms may be used instead of attenuation.

In order to establish a "decay curve", the decay values of the biological sample are determined at least for one, but preferably for a plurality of time points with respect to a specific reference point, e.g. the starting point of the coagulation process. The attenuation values are then plotted over time. Typical decay curves of plasma samples are characterized by their "S" shape or sigmoid curve progression, respectively. Alternatively, the attenuation is measured only at two time points, namely the starting point of the coagulation process and the end stage of coagulation after (near) complete development of the coagulation process.

According to the present invention, "characteristic value" refers to such information that may be extracted from the attenuation curve, for example, by using common mathematical methods, such as differentiation, derivative, integration, etc., which is indicative of the fibrinogen concentration in the biological sample.

When a function of fibrinogen to which such characteristic values are added is recorded, according to the present invention, a "fitting function" can be calculated from the recorded characteristic values. Such a procedure is referred to as "curve fitting," i.e., the process of constructing a curve or mathematical function that best fits a series of data points. Curve fitting may involve interpolation, where an exact fit to the data is required, or smoothing, where a "smoothing" function is constructed that approximates the fit data. Curve fitting includes regression analysis, which focuses on statistically inferred problems, such as how much uncertainty exists in a curve fitted to observed data with random errors.

According to the invention, according to the fitting functionNumerical "determination" of the initial fibrinogen concentration in a biological sample is accomplished by using methods well known in the art, such as extrapolation, including linear, polynomial, conical, or curvilinear extrapolation. For linear fitting, the following equation may apply: fy ═ α × fg_add+ β, fy being a characteristic value, fg_addFor the added fibrinogen, α and β are fitting parameters, respectively the slope and offset of the linear fit. The fibrinogen concentration may then be fg_Sample(s)＝-β/α。

In the device according to the invention, by "container" is meant any receptacle suitable for receiving a biological sample and fibrinogen, such as a well, a cuvette, a kit, a test tube or the like, possibly divided into a plurality of sub-containers.

The "measurement unit" comprises any measurement means suitable for measuring the attenuation of said biological sample, such as a photometer.

"computing unit" refers to a device comprising hardware and software components for processing information, e.g. a processor, a computer, e.g. a program, an algorithm for a computer, etc.

Instead of a decay curve in all embodiments of the invention, a "mass/length curve" may be used. Mass/length is the average mass of the average fibrin fiber divided by its length. The mass/length curve and the attenuation/optical density/turbidity curve are related to each other by an equation extracted from the physical properties of the fibrin fiber. Here, the relation between turbidity and average mass/length ratio is exemplarily shown:

wherein τ is the haze which is the negative natural logarithm of the transmittance T, N_ATo convert the density into Da/cm³Is the average mass/length ratio of the fiber in daltons per centimeter, and a and B are lumped parameters that can be determined in independent experiments or by measuring fixed mass/length ratio optical densities of known solute concentrations at different wavelengths. In the preferred embodimentIn an embodiment, the values of A and B at a wavelength of 632.8nm may be 6.76 χ 10, respectively²²And 1.41 χ 10²⁴. The relationship between attenuation and/or optical density and mass/length ratio directly follows this equation.

In this alternative, the characteristic value indicative of the fibrinogen concentration in the biological sample is similarly extracted from the mass/length curve. For example, the maximum ratio observed in a sample with fibrinogen added indicates a linear relationship between the fibrinogen added and the maximum ratio. This relationship can similarly be used to infer the initial fibrinogen concentration of the sample. The feature values extracted from the average quality/length ratio related to the dynamics of the curve can be optimally approximated by a reciprocal function or alternatively by a power or exponential function.

According to a further development of the method according to the invention, said step (7) is realized by the following steps:

(7.1) extrapolating the fitting function of step (6) to the value of added fibrinogen where the characteristic value is zero to obtain an extrapolated value;

and/or

Extrapolating the fitted function of step (6) to the value of added fibrinogen where it coincides with a hypothetical sample having a fibrinogen level of zero to obtain an extrapolated value;

(7.2) determining an initial fibrinogen concentration in the biological sample by multiplying the extrapolated value by-1.

According to the present invention, the "value" of added fibrinogen may refer to any measure reflecting the added fibrinogen, e.g. the concentration of fibrinogen, e.g. expressed in g/L, or the amount of fibrinogen added, etc.

According to the present invention, a "hypothetical sample" refers to a "theoretical sample" that is not an actually existing sample but is a result of a mathematical operation.

Such an embodiment provides reliable results in case a linear fit has been calculated. The linear fit is extrapolated to the intersection with the horizontal axis, which corresponds to the characteristic value in the case where fibrinogen is not present in the sample, this value at the intersection indicating the fibrinogen concentration that needs to be added in order to obtain a zero concentration of fibrinogen in the sample. Therefore, this intersection will be negative (or zero) and must be multiplied by-1 to obtain the fibrinogen concentration or level in the initial sample.

In a further development, the computer program of the invention therefore comprises program code means for causing a computer to carry out at least the steps (4), (5), (6), (7), (7.1) and (7.2) of the method according to the invention when said computer program is carried out on the computer.

In a further development, therefore, a computer-readable non-transitory storage medium contains instructions for execution by a processor, wherein the instructions cause the processor to perform at least steps (4), (5), (6), (7), (7.1) and (7.2) of the method according to the invention.

According to a further development of the method according to the invention, the characteristic value indicative of the fibrinogen concentration in the biological sample is selected from the group consisting of:

a difference in attenuation of the biological sample between a first time point and a second time point (delta attenuation), preferably a difference in attenuation of the biological sample between a start of a coagulation process as the first time point and an end of a coagulation process as the second time point (delta attenuation);

maximum decay rate (time derivative of decay curve);

features corresponding to time, including: lag time, time to reach (a certain degree of) maximum attenuation.

According to the present invention, "lag time" refers to the point in time when attenuation begins to increase rapidly due to the formation of fibrin fibers thick enough to significantly scatter incident photons. The lag time may be found, for example, by finding the intersection of the tangent at the point of maximum velocity with the horizontal line of the attenuation at the start of the coagulation process, or the point in time at which the second derivative of the attenuation curve with respect to time is at its maximum or with the attenuation value (e.g. 0.1).

It has been demonstrated that all such characteristic values are indicative of the fibrinogen concentration in the biological sample. Delta attenuation is a preferred feature extracted from the attenuation curve, which is exemplary used to demonstrate the present implementation. In case delta attenuation is used as characteristic value, the attenuation will be measured at least two points in time, for example, after the coagulation process has started and has been completed, i.e. when the coagulation has (nearly) fully developed. The time span between two time points may vary, for example, depending on the trigger used to start the coagulation process. A preferred time span may be 1 to 1000 seconds, further preferably 10 to 500 seconds, and highly preferably 100 to 200 seconds, especially in the case of using 1NHI trombone (1NIH trombine) U/mL as a coagulation trigger.

According to another aspect, the fitting function of step (6) is selected from the following options:

a linear function;

a non-linear function comprising: exponential function, power function, reciprocal function.

Any of such functions may be preferred in fitting the extracted feature values according to the nature and behavior of the respective features. As the inventors were able to achieve, a linear function could be used to fit the delta attenuation plotted against fibrinogen. The inventors have also realized that the features corresponding to time may be fitted with a non-linear function. The latter feature tends to infinity as the fibrinogen concentration in the sample tends to 0. 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 the fitted form α/(fg)_addβ) (which is preferred if the features are correlated with points in time), wherein fg_addFor the concentration of fibrinogen added, α, β are unknown parameters fitted with an algorithm, - β equals the fibrinogen level of the sample, when the vertical asymptote is found at β. Another characteristic, i.e. the maximum rate of the decay curve, is preferably used in the form α × (fg)_add–β)²Fitting a function of (1), wherein fg_addAlpha, beta are unknown positive and negative parameters, respectively, fitted using an algorithm for the concentration of fibrinogen added. In the case where the minimum of the quadrant function can be found at β, the reason is thatThis- β is also equal to the fibrinogen level of the sample.

According to another aspect in step (2), the coagulation process is initiated by adding a coagulation trigger to the biological sample, the coagulation trigger preferably being selected from the group comprising:

an active coagulation factor comprising: thrombin (F2a), Tissue Factor (TF), active fx (fxa), active procoagulant (FVIIa);

a snake venom thrombin-like enzyme comprising: batroxobin and reptilase;

kaolinite, micronized silica, ellagic acid.

The advantage of this measure is that the coagulation process is preferably effectively initiated by providing a coagulation trigger of the kind that has proven to be particularly suitable for the present invention. Although the coagulation triggers thrombin (F2a) and the snake venom thrombin-like enzymes are preferred, all of the mentioned coagulation triggers are suitable initiators of the coagulation process, providing reliable results.

Preferably, the coagulation trigger is added to the biological sample in a sufficiently high concentration such that the shape of the decay curve is substantially determined by the concentration of fibrinogen in the biological sample. The amount of fibrinogen in the sample has a large influence on the shape of the decay curve, and the coagulation assay of step (3) may be developed in such a way that in case of sufficiently high concentrations of thrombin or another coagulation trigger with similar activity, the difference between the decay curves of the biological sample is (substantially or only) due to its difference in fibrinogen concentration. A thrombin concentration of greater than or equal to 1NIH U/mL is reasonable, with higher values of thrombin concentration being more preferred, as this will shorten assay time and will reduce the effect of possible feedback in the system. For the snake venom thrombin-like enzyme, a concentration of 1BU/mL or higher is reasonable, and for the coagulation factor, a concentration higher than 10nM is preferred.

In another aspect of the method according to the invention, in step (1), the biological sample is provided in at least two aliquots, allowing to perform steps (2) to (4) in said at least two aliquots, preferably in parallel, so as to obtain at least two decay curves.

Even if multiple predetermined fibrinogen concentrations can be added to a single aliquot of a biological sample, dividing it into several aliquots, adding different predetermined fibrinogen concentrations to each of them can yield more reliable results. The terms "at least two equal parts" and "at least two attenuation curves" include even more equal parts and attenuation curves of 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 1000, etc., respectively. In a preferred embodiment, the biological sample may be provided in a plate or kit, including a plurality of wells or chambers. Each of the wells or chambers may contain a known fibrinogen content.

Suitably, in another aspect of the device according to the invention, the container is a multi-well container, preferably a microtiter plate, capable of receiving at least two aliquots of the biological sample.

Such multi-well vessels or microtiter plates allow the application of the method according to the invention on a large or industrial scale.

In a further aspect of the inventive device, the measuring unit is an optical measuring system comprising at least one light source and at least one light detector.

The advantage of this measure is that such a measuring unit is provided which is particularly suitable for working in connection with the present invention. It goes without saying that the optical measurement system may have 2, 3, 4, 5 and more light sources and detectors, for example, depending on the number of aliquots of biological sample and the specific technical requirements of the device.

As mentioned above, another subject of the present invention is the use of fibrinogen added to a biological sample in at least one predetermined concentration to determine the initial fibrinogen concentration in said biological sample.

The inventors have realized that in order to determine the initial concentration of fibrinogen in a biological sample, it is not necessary to construct an external calibration curve from several plasmas with known fibrinogen levels. Instead, an internal calibration curve may be used, as this has been achieved by the present invention. Such use can be easily automated, for example, in the form of a POC platform with multiple chambers of known fibrinogen content, thus eliminating the need to use calibration plasma.

It shall be understood that preferred embodiments of the invention may also be any combination of the dependent claims with the respective independent claims.

These and other aspects of the invention are apparent from and will be elucidated with reference to the embodiments described hereinafter.

Drawings

In the drawings:

figure 1 shows a typical example of attenuation (on the vertical axis) or change in optical density or turbidity measured over time (on the horizontal axis) in thrombin-initiated clotting plasma. After a short lag time in converting fibrinogen to fibrin, the activated fibrin monomers begin to polymerize and form coarse fibrin fibers that cause light scattering, which can be detected by a decrease in transmitted light or an increase in turbidity or attenuation;

fig. 2 shows the delta attenuation in the samples and the samples with 0.5, 1, 1.5, 2g/L fibrinogen added (open circles). A linear function may be used to fit the measurement points. Extrapolating the linear fit to the intersection with the horizontal axis (corresponding to delta attenuation without fibrinogen in the sample), indicating an initial fibrinogen concentration of 3.14159g/L for the sample;

FIG. 3 shows the delta attenuation observed in fibrinogen deficient plasma with the addition of 1, 2, 3 and 4g/L of fibrinogen and triggered with 0.02(o), 0.06(+) and 1(x) NIH U/mL. Using the methods described herein, the fibrinogen concentration in the samples was calculated using the fitting parameters of the linear function, yielding fibrinogen at 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, respectively;

FIG. 4 shows the change in lag time in seconds observed with a simulated fibrinogen addition test using samples containing 3.14159g/L fibrinogen ranging from-3.1 (corresponding to fibrinogen extraction) to 0.5 g/L. The finding is in the form of lag time (fg)_add)＝85.7234/(fg_add-3.1416) to closely fit the observations. A vertical asymptote is found at-3.1416,

fig. 5 schematically shows a corresponding flow chart of an exemplary embodiment of a method according to the present invention.

Detailed Description

Coagulation of plasma may be initiated by, for example, tissue factor, kaolin, thrombin, or another coagulation agent or trigger, respectively. During such coagulation, the opacity of the plasma sample changes. This results in a reduction in light transmission, which can be measured by the change in sample attenuation, optical density or turbidity over time (Carr and Gabriel, Macromolecules (1980)13(6): 1473-. The amount of fibrinogen in the sample has a high impact on the shape of the attenuation, optical density or turbidity curve, and the coagulation assay can be developed such that at sufficiently high concentrations of thrombin or another similar enzyme with similar activity to fibrinogen, the difference between the attenuation or optical density curves of the plasma sample is primarily due to the difference in fibrinogen concentration. Referring to fig. 1, an example of a typical decay curve for a plasma sample that has been initiated with thrombin. Thus, the features extracted from the attenuation curve are indicative of the fibrinogen level in the sample and can be used to construct a calibration curve using the addition of known fibrinogen levels to the sample.

In the method according to the invention, the calibration by the sample of interest itself is performed by preferably adding at least one, but preferably several, known fibrinogen concentrations to the initial sample instead of a calibration curve based on external control plasma with known fibrinogen concentrations. Interesting features from the attenuation curve are extracted from multiple measurements.

Figure 2 shows the theoretical difference in sample attenuation (calculated in the model) between the start and end of the measurement, also known as delta attenuation, as a function of the concentration of fibrinogen added (here fibrinogen in grams added per litre of plasma sample). Delta attenuation is a preferred feature from the attenuation curve used exemplarily in the present invention. As can be seen in fig. 2, this characteristic varies linearly with the addition of fibrinogen. If there is no fibrinogen in the sample before any fibrinogen is added, there will be no attenuation at zero added fibrinogen because fibrin will not be formed. The non-zero value of the delta attenuation at zero added fibrinogen indicates the initial concentration of fibrinogen in the sample, i.e. the value to be determined by the method according to the invention.

Coagulation characteristics, such as delta attenuation, are measured at zero and one or more non-zero values for the added fibrinogen, thus followed by a step involving calculation of a fit through a plurality of observed characteristic values, sometimes referred to as regression. Extrapolation of the fitted curve then yields the initial fibrinogen concentration in the plasma sample. For the delta attenuation feature, this works as follows:

estimating a linear fit (Δ attenuation ═ α × fg)_add+ β, wherein, fg_addFor added fibrinogen, α and β are fitting parameters, respectively slope and offset of the linear fit);

the fitted function is extrapolated to the value of added fibrinogen where the delta decays to zero. This will be a negative value (or zero);

fibrinogen levels in the samples were calculated as extrapolated values minus. For the linear relationship of the added fibrinogen followed by the delta attenuation, this is equal to the deviation of the straight line divided by the slope (fg)_Sample(s) ＝-β/α)。

The above method with delta attenuation as an observed feature was applied to fibrinogen deficient plasma samples, i.e. such samples where the level of fibrinogen is close to zero. In this example, the sample was found to have a residual fibrinogen antigen concentration of 0.05g/L (detected by ELISA). Next, clotting in fibrinogen deficient plasma was initiated using various thrombin concentrations (0.02, 0.06, and 1NIH U/mL) in combination with various fibrinogen additions (1, 2, 3, and 4 g/L). For each of the (12) test combinations, the turbidity or attenuation of each combination was measured in four replicates. The attenuation curves were averaged and delta attenuation was calculated for each experimental condition, see fig. 3.

The next step is to fit a linear function to each of the aforementioned thrombin concentrations (see figure 2). Next, the fibrinogen concentration of the initial sample is inferred using the equation previously described. This yielded fibrinogen levels 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, which corresponded well to the expected fibrinogen concentrations for 0.02, 0.06 and 1NIH U/mL thrombin, respectively. The best results, i.e. the results closest to the actual fibrinogen level determined by ELISA and the minimum confidence interval, were obtained with 1NIH U/mL thrombin, probably due to the minimal effect of feedback in the coagulation system on the coagulation process.

Alternatively, a similar approach may be followed for other extracted features of the decay curve that may exhibit non-linear behavior for multiple fibrinogen additions. For example, the characteristics corresponding to the time to reach the maximum decay (95%), e.g., the lag time, the time to reach the maximum rate (i.e., the time at which the maximum of the time derivative of the curve occurs) may be fitted to an exponential, power or reciprocal function, e.g., with added fibrinogen as a variable. Such a feature tends to infinity when the true fibrinogen concentration in the sample tends to zero, as seen in experiments simulated using a 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 the fitted form α/(fg)_addβ) (which is preferred if the features are correlated with points in time), wherein fg_addFor the added fibrinogen level, α, β are unknown parameters that are fitted using an algorithm, - β is equal to the fibrinogen level of the sample, when a vertical asymptote is found at β. Another characteristic, i.e. the maximum rate of the decay curve, is preferably in the form of a (fg)_add-β)²Fitting a function of, fg_addAlpha, beta are unknown parameters fitted using an algorithm for the level of fibrinogen added. In case the minimum of the quadrant function can be found at β, therefore- β is also equal to the fibrinogen level of the sample.

Alternatively, features extracted from the average mass/length curve may be used to infer fibrinogen concentration. For example, the maximum rate observed in a sample to which fibrinogen is added indicates that there is a linear relationship between the fibrinogen added and the maximum rate (i.e., the maximum of the time derivative of the curve). This relationship can also be used to infer the fibrinogen concentration of the sample. The features extracted from the average mass/length ratio related to the curve dynamics, i.e. lag time, time to reach maximum rate (i.e. maximum of the curve time derivative), time to reach maximum mass/length ratio (95%) can be best approximated by an inverse or power or exponential function. As previously described, the fibrinogen level of the sample can be inferred by finding a (vertical) asymptote.

Fig. 5 schematically shows a corresponding flow chart of an embodiment of the method according to the invention. In step (S1), a biological sample, such as a plasma sample of an individual suspected of having an altered fibrinogen concentration, is provided. In step (S2), a clotting process is initiated in the biological sample, e.g. by adding a clotting trigger, e.g. thrombin (F2a) or a snake venom thrombin-like enzyme, respectively. In step (S3), fibrinogen is added to the biological sample at least one predetermined concentration. Preferably, multiple amounts of fibrinogen are added to aliquots of the sample in parallel assays, e.g., divided into kits having multiple detection chambers. In step (S4), the attenuation (-S) of the biological sample (S) containing the concentration of added fibrinogen (S) as a function of time is determined and an attenuation curve/curves is/are obtained.

In step (S5), a characteristic value indicative of the fibrinogen concentration in the biological sample, e.g., delta attenuation, is extracted from the attenuation curve and recorded as a function of the fibrinogen added in step (S3). In step (S6), a fitting function is calculated from the feature values recorded in step (S5). In step (S7.1), the fitting function of step (S6) is extrapolated to the value of the added fibrinogen when the characteristic value is zero, or to the value of the vertical asymptote if the characteristic is related to the timing of the coagulation process, or to the value where the quadratic fitting function has a minimum in the case of the maximum rate of the decay curve, to obtain an extrapolated value. In step (7.2), the initial fibrinogen concentration in the biological sample is determined by multiplying the extrapolated value by-1.

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 exemplary 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.

In the claims, the word "comprising" does not exclude other elements or steps, and the word "a" or "an" does not exclude a plurality. A single processor 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.