Method for creating a database for determining light transmission aggregometry reference values and method and device for performing measurements

Abstract

A method for creating a database for determining virtual reference values for light transmission aggregometry measurements, the method comprising the following steps: a. providing platelet-rich plasma "PRP" (17) of a reference blood sample; b. performing light transmission measurements on the PRP (17) of the reference blood sample using a first light wavelength and a second light wavelength different from the first light wavelength; c. providing platelet-poor plasma "PPP" of the reference blood sample; d. performing light transmission measurements on the PPP for determining a PPP reference value; e. assigning the measurement result of step d to the measurement result of step b in the database; f. repeating steps a to e for a plurality of reference blood samples. The database obtained by this method enables determination of virtual reference values, which represent excellent estimates of the PPP reference value. After the database has been created, virtual reference values for the blood sample to be examined can be determined, and LTA measurements can be performed using the virtual reference values without obtaining the PPP of the blood sample to be examined.

Description

Technical Field

[0001] The subject of the present invention is a method for creating a database that can be used to determine a virtual reference value for a light transmission aggregometry measurement. The subject of the present invention is also a method for determining a virtual reference value for performing a light transmission aggregometry measurement on platelet-rich plasma of a blood sample to be examined using the created database, and for performing a light transmission aggregometry measurement using this virtual reference value. Background Art

[0002] In order to evaluate the functionality of thrombocytes (platelets), light transmission aggregation assay (LTA) is one of the most commonly used methods. In order to perform the LTA measurement, so-called platelet-rich plasma (PRP) is obtained from a blood sample provided by a subject by means of centrifugation. Due to the disc-shaped shape with a diameter between 1.5 μm and 3 μm, light scattering occurs at the thrombocytes, which makes the PRP appear as a turbid liquid. After adding an activator to the PRP sample, the thrombocytes cross-link and form aggregates, whereby the light transmittance of the PRP sample (at least in the range where the PRP sample is derived from a healthy subject) increases, and over time, the transmittance approaches a maximum value. In the scope of the LTA measurement, the increase in transmittance (or the decrease in extinction) is measured over time under standardized conditions by directing light at the PRP sample and measuring the intensity of the light emitted from the sample. The document “SAKAYORI TASKUKU et al.: “Evaluation of the Newly Developed Adenosine Diphosphate induced Platelet Aggregation Level System in Aggregometer on Automated Coagulation Analyzer”, CLINICAL LABORATORY, Bd. 65, No. 12 / 2019, December 1, 2019 (2019-12-01), XP 055871304” discloses an LTA method in which the so-called “ADP-induced platelet aggregation level (APAL)” is used to evaluate the LTA measurement. In “LING LI-QIN et al.: “Evaluation of an automated light transmission aggregometry”, PLATELETS (LONDON), Bd. 28, No. 7, 2. February 2017 (2017-02-02), pp. 712-719, XP 055871233” describes a study on the performance of a coagulation analyzer.An overview of new developments in the field of LTA measurement methods is given in the document “LE BLANC JESSICA et al.: “Advances in Platelet Function Tenting-Light Transmission Aggregometry and Beyond”, JOURNAL OF CLINICAL MEDICINE, Bd.9, Nr.8, August 13, 2020 (2020-08-13), page 2636, XP 055871250”. US 2017 / 248576A1 discloses an LTA analyzer having three light sources for emitting three different light wavelengths, wherein different wavelengths are used for different measurement tasks. The document “Mukaide Kae: “Overview of the Automated Coagulation Analyzer CS5100′, Sys-mex Journal International, 2013, XP 055871951” provides a product overview of the automatic coagulation analyzer “CS-5100”.

[0003] In order to be able to interpret changes in light transmission, in the prior art it was necessary to provide a platelet-poor plasma ("PPP") sample from the same subject in addition to the PRP sample. The transmittance of this PPP sample was also determined and used as a reference value for the LTA measurement performed on the PRP. Since the PPP has only a low concentration of thrombocytes, it is usually a transparent liquid and has a maximum transmittance. The transmittance measured at the PRP is usually proportional to this maximum transmittance. Only this behavior makes it possible to meaningfully interpret the LTA measurements in the prior art and in particular to evaluate the maximum degree of aggregation and the speed of the aggregation change.

[0004] The need to draw additional blood to obtain a PPP sample can be burdensome for the subject. This applies in particular to newborns or infants, who naturally have a low blood volume, or to patients whose blood can only be drawn to a limited extent due to pre-existing medical conditions. Furthermore, providing a PPP sample requires additional labor and consumables. Summary of the Invention

[0005] Against this background, the present invention is based on the object of providing a method for creating a database for determining virtual reference values for LTA measurements, a method for determining virtual reference values for performing LTA measurements using the created database, and a method for performing LTA measurements that provide reliable results with minimal effort. This object is achieved by means of the features of the independent claims. Advantageous embodiments are described in the dependent claims.

[0006] The method according to the invention for creating a database for determining virtual reference values for LTA measurements comprises the following steps:

[0007] a. Provide platelet-rich plasma (PRP) as a reference blood sample;

[0008] b. performing light transmission measurement of the reference blood sample PRP using a first light wavelength and a second light wavelength different from the first light wavelength;

[0009] c. providing platelet-poor plasma (PPP) of the reference blood sample;

[0010] d. Performing light transmission measurement on PPP for determining a PPP reference value;

[0011] e. Assigning the measurement result of step d to the measurement result of step b in the database;

[0012] f. Repeat steps a to e for multiple reference blood samples.

[0013] First, some of the terms used within this description will be explained. The method according to the present invention requires the provision of platelet-rich and platelet-poor plasma from a blood sample. This can be done in a manner known in principle, such as described in the document "Leitlinie-Thrombozytopathien, Version 2.1 (AWMF Register No. 086-003, update 2 / 2018)." In particular, a defined amount of a suitable anticoagulant (e.g., sodium citrate) is added to the blood sample to prevent blood clotting.

[0014] When performing a light transmission measurement on a PRP or PPP of a blood sample, light of a predetermined intensity is preferably directed onto a volume of the PRP or PPP, typically located in a light-transmitting container, wherein the intensity of the light is measured after passing through the volume. The ratio of the initial predetermined intensity to the intensity after passage yields a light transmittance, which can represent the result of the light transmission measurement. In the case of a light transmission measurement on PPP, the transmittance can form a PPP reference value.

[0015] If light transmission measurements are performed with a first wavelength and with a second wavelength, these measurements can be performed temporally sequentially or also simultaneously for different partial volumes of the examined volume.

[0016] In the context of this description, two wavelengths are considered different if they differ by at least 10 nm. In one embodiment, the first wavelength differs from the second wavelength by at least 50 nm, preferably by at least 100 nm, and more preferably by at least 200 nm. The first wavelength may be in the range of 300 nm to 500 nm, and preferably between 345 nm and 465 nm, and more preferably between 385 nm and 425 nm. Furthermore, the second wavelength may be in the range of 500 nm to 800 nm, and preferably between 550 nm and 700 nm, and more preferably between 600 nm and 640 nm.

[0017] Within the scope of the present invention, light transmission measurements of PRPs can be performed using monochromatic light having at least the aforementioned wavelength interval or within the aforementioned range. It is also possible within the scope of the present invention to use a light beam formed by superimposing multiple light wavelengths for light transmission measurements. In this case, the wavelength of the light beam is given by the average value (preferably weighted by intensity) of the wavelengths contained in the light. The wavelength interval between the two light beams is thus predetermined by the interval between the average values of the light beams. When, within the scope of this description, we talk about "light of one wavelength," this can also refer to the corresponding light beam whose wavelength average value is at this wavelength.

[0018] Within the scope of the present invention, a light transmission measurement is performed on the PRP of a reference blood sample using at least two different wavelengths, from which, in particular, the transmittance can be determined. Additionally, a reference value for light transmission (hereinafter also referred to as a PPP reference value) is determined for the PPP of the reference blood sample in a manner known in principle from the prior art. Within the scope of the present invention, it has been recognized that there is a statistically significant relationship between the measured values of the PRP obtained using at least two different wavelengths and the PPP reference value, which relationship is revealed when the values stored in a database are evaluated for multiple reference blood samples. This statistical relationship makes it possible, after the database has been created, to use it to determine a virtual reference value for other blood samples to be examined from the patient, which represents a reliable estimate of the PPP reference value, without having to obtain and measure additional PPP samples from the same patient. For example, a mathematical relationship between the measured values obtained for the PRP of the blood sample to be examined and the virtual reference value can be determined from the database. This is explained in more detail below in conjunction with the method for determining the virtual reference value and the method for performing the LTA measurement. Alternatively, the virtual reference value for the PPP reference value can also be determined from the database using other means, in particular by means of statistical methods known in principle (if necessary with the assistance of artificial intelligence). Such methods are generally known to those skilled in the art. The core of the present invention lies in the knowledge that the method steps according to the present invention can create a database containing sufficient information for determining a virtual reference value. Due to the database according to the present invention, additional blood sampling for obtaining a PPP sample and the associated expense can be avoided.

[0019] Within the scope of the present invention, it has been recognized that for a specific blood sample, not only the PPP reference value but also the measured values of PRP obtained using different wavelengths are related to the physiological state of the blood sample, that is, in particular the type and concentration of components contained in the blood sample, which influence the transmittance of the PPP sample (and thus the PPP reference value) and the PRP sample. Such components can be, for example, hemoglobin, ceruloplasmin, bilirubin, lipoproteins or other components delivered by the drug. The inventors assumed that there are specific components that influence the transmittance depending on the wavelength of light. Therefore, by using at least two different wavelengths of light, a correlation is obtained in the database between the measured values determined for PRP and the PPP reference value, because not only the PRP measured value but also the PPP reference value are influenced in a predetermined manner by the type and / or concentration of components contained in the blood sample. After the database has been created, when examining other blood samples, the PPP reference value can be inferred simply by measuring the PRP using different wavelengths and, if necessary, using the database (or the mathematical relationship obtained therefrom), without having to obtain additional PPP samples.

[0020] In a preferred embodiment, the method for creating a database comprises the further steps of:

[0021] g. After performing the measurement according to step b, adding a predetermined amount of activator to the reference blood sample PRP;

[0022] h. repeating the measurement according to step b after adding the activator and before the start of aggregation triggered by the activator;

[0023] i. Assign the measurement result of step h to the measurement result of step d in the database;

[0024] .j. Repeat steps g to i for multiple reference blood samples.

[0025] Here, the term "activator" refers to one or more reagents that are set up to trigger the aggregation of thrombocytes after being added to PRP. The activator may include one or more reagents selected from ristocetin, arachidonic acid, adenosine triphosphate (ADP), adrenal hormone (epinephrine), collagen, thrombin receptor activating peptide (TRAP). After the addition of the activator, aggregation begins with a time delay. The thrombocytes remain in an activated state for a certain period of time, and there is still no increase in light transmission, which is triggered by the cross-linking of the thrombocytes. Preferably, repeated measurements of step h are performed during this time period, i.e. before actual aggregation occurs. The light transmission measurement according to step h after adding the activator is preferably performed after adding the activator within a time period between 0 and 10s, preferably between 0 and 5s, wherein a time average is preferably formed over a time period between 1s and 6s, preferably between 3s and 5s, in order to detect the transmission value. The light transmission measurement before the addition of the activator preferably takes place immediately before the addition of the activator, for example, between 0 and 10 seconds before the addition of the activator. The mixing ratio between the volume of PRP and the volume of activator added to the PRP volume according to step g is typically 9:1, but can also be, for example, between 20:1 and 2:1, preferably between 15:1 and 4:1, and more preferably between 7:1 and 11:1.

[0026] In the case of the above-described embodiment, light transmission measurements are performed using two light wavelengths not only before but also after the addition of a predetermined amount of activator. It has been shown that the PPP reference value can be inferred more accurately and more convincingly by means of the additionally acquired measurement values. In particular, it has been recognized that the addition of a predetermined amount of activator to the PRP causes an initial and individual PRP sample-related change in light transmission, which can be a decrease or an increase in light transmission. This initial change also has a dependency on the physiological state of the blood sample and in particular on its components. In the described embodiment, additional information is therefore added to the database, which additional information makes it possible to statistically infer the PPP reference value in an improved manner.

[0027] In one embodiment of the present invention, in method step g, a first activator is used for the first reference blood sample, and a second activator, different from the first activator, is used for the second reference blood sample. This makes it possible to distinguish between measurement data obtained using different activators within the database. If a virtual reference value for the blood sample to be examined is later determined using a specific activator, the measurement results (or the mathematical relationships determined therefrom) obtained for the reference blood sample using the same activator can be used within the database. If it is expected that different activators do not lead to different measurement results, the measurement results obtained using different activators can also be combined in the database or used together to determine the mathematical relationship.

[0028] The light transmission measurement of step b and / or step h can be performed using at least three different wavelengths of light. In particular, the light of the first wavelength can have a wavelength in the range between 380 nm and 420 nm, preferably between 400 nm and 410 nm. The light of the second wavelength can have a wavelength in the range between 500 nm and 550 nm, preferably between 520 nm and 530 nm. The light of the third wavelength can have a wavelength in the range between 620 nm and 700 nm, preferably between 620 nm and 630 nm. It has been shown that the accuracy is further improved by using three wavelengths, and the PPP reference value can be deduced with said accuracy. Preferably, the three wavelengths are used not only for the measurement before the addition but also for the measurement after the addition.

[0029] In an advantageous embodiment, the plurality of reference blood samples comprises a first reference blood sample and a second reference blood sample, wherein the first reference blood sample comprises at least one first component that is not present in the second reference blood sample or is present in the second reference blood sample at a lower concentration. For example, the concentration of the component in the first reference blood sample may be more than 1.5 times, preferably more than 3 times, and more preferably more than 5 times, the concentration of the component in the second reference blood sample. A concentration difference of, for example, 20 times is also possible. The first component may preferably be selected from the group consisting of hemoglobin, ceruloplasmin, lipoproteins, triglycerides, and bilirubin. The first component may also be a dye or component from a drug or food, or a decomposition product thereof. If the reference blood samples differ in their components, the database covers a broader base of different physiological states of the blood samples. Preferably, the database is created using a plurality of reference blood samples (e.g., more than 5, preferably more than 10, and more preferably more than 20), wherein the reference blood samples differ from each other pairwise in the type and / or concentration of at least one component, for example due to the aforementioned concentration differences. It is also possible to create a database using reference blood samples that differ from one another pairwise by more than one, preferably more than two, and more preferably more than five components, wherein the differences can be given by the above-mentioned concentration differences. This further improves the accuracy of the statistical inferences that can be made from the database.

[0030] It can be provided that the PRP volume of the reference blood sample is divided into a plurality of subvolumes, wherein the method according to the invention is performed on each of these subvolumes, wherein the determined measured values are then averaged over the plurality of subvolumes and recorded in a database. This averaging can further increase the statistical accuracy.

[0031] In one embodiment, the first component is added manually to the first reference blood sample. It is also possible to feed the component to different reference blood samples at different predefined concentrations. This has the advantage that the influence of the component on the measured values determined within the scope of the present invention can be systematically detected and recorded in a database. In particular, a measurement series can be performed using multiple reference blood samples, wherein the component is added to different reference blood samples at multiple different concentrations and / or wherein multiple different components are added to different reference blood samples, if necessary at different concentrations.

[0032] In principle, it is also possible that at least some of the reference blood samples used for carrying out the method according to the invention are obtained from subjects, wherein the reference blood samples may differ from one another in the type and / or concentration of components contained therein, for example due to a prior illness or medication or other influences.

[0033] The subject matter of the present invention is also a method for determining a virtual reference value for performing an LTA measurement of the PRP of a blood sample to be examined, using the database according to the present invention. The method comprises the following steps:

[0034] a. Provide PRP of blood sample to be examined;

[0035] b. performing light transmission measurement of the PRP of the blood sample to be examined using a first light wavelength and a second light wavelength different from the first light wavelength;

[0036] c. Determine a virtual reference value for the blood sample to be examined using the measurement results obtained by step b and the database according to the invention.

[0037] After creating the database according to the present invention, the above-described method can be used to determine so-called virtual reference values for other blood samples to be examined. These virtual reference values represent estimates of the PPP reference values determined based on the database and the PRP measurements performed. Using these virtual reference values, subsequent LTA measurements can be meaningfully interpreted without having to obtain a PPP sample from the same patient.

[0038] The method for determining a virtual reference value preferably comprises the further steps of:

[0039] d. After the measurement according to step b is performed, a predetermined amount of activator is added to the PRP of the blood sample to be examined;

[0040] e. After adding the activator to the PRP of the blood sample to be examined and before starting the aggregation triggered by the activator, repeating the measurement according to step b;

[0041] f. Including the measurement results obtained in step e in the determination of step c.

[0042] The provision of the PRP according to step a and the execution of the light transmission measurement according to steps b and / or e can be carried out in the same manner as described above in conjunction with the method for creating a database. In particular, it can be provided that for the measurement, the (at least substantially) same light wavelength or light wavelength range is used and / or the (at least substantially) same ratio between the volume of the PRP and the volume of the added activator is used. In this regard, the method for determining the virtual reference value can be improved by the features already described above in conjunction with the method for creating a database. This ensures that the measurement results are detected under conditions that are the same or at least as similar as possible to those used when creating the database. This makes it possible to make inferences based on the measurement results of the blood sample to be examined with high precision.

[0043] In the simplest case, the virtual reference value can be determined by comparing the measurement results of the blood sample to be examined (obtained in steps b and, if necessary, e) with the measurement results contained in the database. If the database contains measurement results of a reference blood sample that are identical or substantially identical to the measurement results of the blood sample to be examined, the PPP reference value present in the database for this reference blood sample can be used as the virtual reference value.

[0044] However, in many cases, no data records of the reference blood sample will be found in the database that fully correspond to the data records of the blood sample to be examined. Furthermore, individual data records contained in the database may also be subject to measurement errors, making the retrieval of a single data record to determine the virtual reference value potentially error-prone. Therefore, it is preferably provided that a mathematical relationship based on the database is constructed, which has the measurement results obtained from the blood sample to be examined as input variables and the virtual reference value of the blood sample to be examined as output variable. In particular, it has been shown that the virtual reference value virtPPP (also referred to as virtREF in some parts of the description) can be expressed as a mathematical function of the measured value:

[0045] virtPPP=virtPPP(xd1, xd2, xd3, xd4), where

[0046] xd1: transmittance measured on the PRP of the blood sample to be examined at wavelength λ1 before addition of the activator,

[0047] xd2: transmittance measured on the PRP of the blood sample to be examined at wavelength λ2 before addition of the activator,

[0048] xd3: transmittance measured on the PRP of the blood sample to be examined at wavelength λ1 after addition of the activator,

[0049] xd4: transmittance measured on the PRP of the blood sample to be examined at wavelength λ2 after addition of the activator,

[0050] λ1: first wavelength, and

[0051] λ2: second wavelength.

[0052] The mathematical relationship or function virtPPP(xd1, xd2, xd3, xd4) can be obtained from a database by means of statistical adaptation methods known in principle from the prior art.

[0053] In an advantageous specific embodiment, the ratios V1 =xd1 / xd2 , V2 =xd3 / xd4 and V3 =V2 / V1 are determined within the scope of the mathematical relationships and used to calculate the virtual reference value.

[0054] If more than two light wavelengths are used to create the database and measure the blood sample to be examined, the function can be correspondingly expanded by means of statistical adaptation methods known in principle from the prior art.

[0055] The subject matter of the present invention is also a method for carrying out an LTA measurement on a blood sample to be examined, comprising the following steps:

[0056] a. performing a method for determining a virtual reference value of a blood sample to be examined according to the present invention,

[0057] b. Perform LTA measurement using a virtual reference value.

[0058] In particular, the light transmission aggregometry measurement can follow the method for determining the virtual reference value over time. If an activator has already been added when determining the reference value, the aggregation caused thereby can be detected within the scope of the subsequent LTA measurement. The activator is preferably designed so that it is suitable for LTA measurement. The LTA measurement after the method for determining the virtual reference value can in principle be carried out with a single wavelength or with multiple wavelengths, which are guided alternately through the volume of the PRP or simultaneously through different sub-volumes of the PRP.

[0059] The subject matter of the present invention is also a device for determining a virtual reference value for performing an LTA measurement of a PRP of a blood sample to be examined, the device comprising an illumination module for selectively emitting light of a first light wavelength and a second light wavelength different from the first light wavelength; a sample receptacle for inserting a PRP sample so that the light of the illumination module passes through the PRP; a light sensor designed to detect the light passing through the PRP; and a control module designed to control the device so that the following steps are performed:

[0060] a. performing light transmission measurement of the PRP of a blood sample to be examined using a first light wavelength and a second light wavelength different from the first light wavelength;

[0061] b. Using the measurement results obtained by step a and the database according to the invention, a virtual reference value for the blood sample to be examined is determined.

[0062] In a preferred embodiment, the device comprises an applicator for automatically adding a predetermined amount of activator to the PRP, wherein the control module is configured to operate the device so as to perform the following steps:

[0063] c. After performing the measurement according to step a, adding a predetermined amount of activator to the PRP of the blood sample to be examined;

[0064] d. after adding the activator to the PRP of the blood sample to be examined and before the aggregation triggered by the activator is started, repeating the measurement according to step a;

[0065] e. Include the measurement results obtained in step d in the measurement of step b.

[0066] The device can in particular include a calculation module that is designed to access a mathematical relationship based on a database according to the present invention and to determine a virtual reference value based on this mathematical relationship and the measured values obtained in step a or in steps a and d. The mathematical relationship can in particular be stored in the calculation module.

[0067] The device according to the invention can be improved by further features already described in connection with the method for creating a database, the method for determining a virtual reference value and / or the method for performing a light transmission aggregometry measurement. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0069] Figure 1 shows exemplary light transmission measurements performed on PRP of three reference blood samples having a first wavelength;

[0070] Figure 2 Show Figure 1 Light transmission measurement, wherein the measurement curve has been shifted to zero at the time point t = -8s;

[0071] Figure 3 Shows the time Figure 2 The ratio between the transmittance shown in and the corresponding PPP reference value of the corresponding reference blood sample;

[0072] Figure 4 shows an exemplary LTA measurement performed on the PRP of a blood sample to be examined;

[0073] Figure 5 Shows the time Figure 4 The ratio between the transmittance shown in and the virtual reference value and to the measured PPP reference value of the blood sample to be examined;

[0074] Figure 6 shows a comparison between the measured PPP reference value and the virtual reference value determined using the method according to the invention for a large number of blood samples to be examined;

[0075] Figure 7 Show the Figure 6 Statistical evaluation of the results shown in ;

[0076] Figure 8A schematic illustration of a device according to the invention for determining a virtual reference value is shown. DETAILED DESCRIPTION

[0077] Figure 1 Three exemplary light transmission measurements performed on PRP of three different reference blood samples are shown over time. The change in the measured value of the intensity measuring device is plotted as a function of time. The measured value is proportional to the decimal logarithm of the transmittance (below, transmittance is denoted by the letter T). Therefore, an increase in the measured value corresponds to an increase in the transmittance. The measurement includes the time period t=-8s to t=6 minutes, wherein at the time point t=0s, the activator adenosine diphosphate (ADP) is added to the respective PRP sample in a volume ratio of 1:9 (one part ADP, 9 parts PRP). The measurement was performed at a wavelength of λ1=625nm. Reference blood samples 1 to 3 differ from each other only in the type of component that was manually added to the respective sample. No component was manually added to reference blood sample 1, while fat emulsion was added to reference blood samples 2 and 3 in a concentration of 75mg / dl or 151mg / dl. In Figure 1 As can be seen in , the addition of fat emulsion results in a smaller absolute value of the transmittance.

[0078] Figure 2 Show Figure 1 Measurement results, where the curve has been shifted to zero to make the changes in transmittance more apparent. It can be seen that shortly after the addition of the activator (between t = 0 and t = 10 s), the transmittance of Reference Sample 1 barely changes or even decreases. During this time period, the activation process of the thrombocytes begins, but aggregation has not yet occurred. After the expiration of the time period, the thrombocytes begin crosslinking at approximately t = 10 s and form aggregates in the PRP, resulting in an increase in transmittance over time. After approximately t = 150 s, the maximum transmittance is reached and remains virtually unchanged thereafter.

[0079] In the case of reference samples 2 and 3, at time point t=0, it can be seen that Δ(log 10 T) = 0.03 and Δ(log 10 Even in the case of these reference blood samples, aggregation starts subsequently (from about t=10 s) and the transmittance increases further.

[0080] The absolute value of the change in transmittance is not convincing in itself, as it depends, for example, on the concentration of thrombocytes contained in the sample and the concentration and type of other components contained therein. For this reason, in the prior art, it is necessary to determine a PPP reference value by performing a light transmission measurement on the PPP of the same blood sample and to correlate the measured values obtained at the PRP with this measured PPP reference value. Such a PPP reference value was determined in the reference blood sample shown above in a manner known in the prior art. Figure 3 The corresponding ratios of the transmittance of reference blood samples 1 to 3 to the corresponding PPP reference values are shown over time. It can be seen that the ratio approaches a value of approximately 85 in all three samples over time. Figure 3 From the measurement data shown in , the functional capacity of the thrombocytes can be inferred.

[0081] The following is based on Figures 1 to 3 The measurement data shown in exemplify the method for creating a database according to the present invention. Figures 1 to 3 In addition to the transmission measurements shown in FIG, light transmission measurements were performed on the PRP of the same reference blood sample at a wavelength of λ2 = 405 nm shortly before the addition of the activator (at t = -3 s) and shortly after the addition of the activator (by forming an average value over the time period between t = 2 s and 6 s). For example, the following measurement results were determined for reference blood sample 1:

[0082] - Transmittance at wavelength λ1 before adding the activator at time t = -3 s: xd1 = 0.82676788;

[0083] - Transmittance at wavelength λ2 before adding the activator at time t = -3 s: xd2 = 0.532220558;

[0084] - the transmittance at wavelength λ1 after addition of the activator, obtained by averaging over the period between t=2s and t=6s: xd3=0.840990463;

[0085] - the transmittance at wavelength λ2 after addition of the activator, obtained by averaging over the period between t=2s and t=6s: xd4=0.551279411, and

[0086] - PPP reference value measured at PPP of the reference blood sample: PPP-Ref = 1.0228.

[0087] The values xd1, xd2, xd3, xd4 and the PPP reference value have been added to the database. Corresponding values have also been determined for reference blood samples 2 and 3 and have also been added to the database. The database is illustrated in Table 1 below.

[0088]

[0089] Table 1

[0090] Corresponding measured values were detected for the other reference blood samples 4 to 7 and also added to the database. Reference blood samples 4 to 7 differ from each other only in the components that were manually added to the PRP. Table 2 below lists the components and their concentrations together with the detected measured values.

[0091]

[0092] Table 2

[0093] Patterns based on the influence of additionally added components can be seen within the database. For example, a comparison of transmission values can reveal that the added components have a specific influence not only on the determined transmission values but also on the measured PPP reference values. For example, a comparison of the xd1 and xd2 values for samples 4 to 6 shows that the transmission change triggered by the addition of bilirubin or hemoglobin is much more pronounced at wavelength λ2 than at wavelength λ1.

[0094] For example, another recognizable pattern is that in the case of sample 1 the addition of the activator causes only a small change between the transmission values xd1 and xd3, whereas in the case of samples 2 and 3 the change (i.e. with increasing fat emulsion concentration) increases significantly (see Figure 2 ). At the same time, the addition of ingredients leads to more or less significant changes in the PPP reference value.

[0095] After the measurements explained above as an example have been carried out on a large number of reference blood samples and have been stored in a database, the above-described type of pattern leads to statistically significant relationships which enable the establishment of a mathematical relationship for determining the virtual reference variable.

[0096] An exemplary mathematical relationship is set forth below:

[0097] Determine the parameters:

[0098] V1=xd1 / xd2

[0099] V2=xd3 / xd4

[0100] V3=V2 / V1

[0101] Ve=(V1+V2) / e V3 and

[0102] X xdPIP =xd4+xd3-xd2-xd1. In addition, the parameters

[0103] X vPIP=(1-xd3)×(-log 10 (xd3))×(V1+Ve) / e Ve Based on the above parameters, the temporary virtual reference parameter 1virtPPP can be determined as follows:

[0104] 1virtPPP=1+X xdPIP -X vPIP .

[0105] In addition, the calculation factor

[0106] F1 = xd3 / 1virtPPP It has been shown that for those PRP samples whose value of the factor F1 is less than a threshold value (in this example F1 < 1.19), the temporary virtual reference parameter 1virtPPP represents a good estimate of the PPP reference value.

[0107] As long as the factor F1 is greater than a threshold value (ie, F1>1.19 in this example), the corrected virtual reference value 2virtPPP is determined as follows:

[0108] 2virtPPP=1.26×F1 -1，134

[0109] For such PRP samples with a factor F1>1.19, the corrected virtual reference value 2virtPPP represents a good estimate of the PPP reference value. Thus, currently, the following reproducible mathematical relationship has been determined from the database for determining the virtual reference value:

[0110]

[0111] The virtual reference values virtPPP determined in this way for reference samples 1 to 6 are shown in the above Tables 1 and 2. It can be seen in the tables that there is a good agreement with the measured PPP reference values (PPP-Ref).

[0112] Figure 4 The LTA measurement of the PRP of the blood sample to be examined is shown. Figure 1 and 2 The measurements shown in Figure 1 were performed under the same measurement conditions between t = -8 s and t = 6 minutes, with the measurement curve at time t = -8 s being shifted to zero. At time t = 0 s, the activator ADP was added to the PRP in a ratio of 9:1. Before and after the addition of the activator, the following transmission values xd1, xd2, xd3, and xd4 were determined in the manner described above for wavelengths λ1 = 625 nm and λ2 = 405 nm:

[0113] xd1=0.698934, xd2=0.257505, xd3=0.726952, xd4=0.280035.

[0114] With the help of the above mathematical relationship, the above-mentioned intermediate values are calculated as follows:

[0115]

[0116] The temporary virtual reference values are:

[0117] 1virtPPP=1.0259.

[0118] Furthermore, factors were identified.

[0119] F1=1.411.

[0120] Since this factor exceeds the value of 1.19, it is derived as a virtual reference value:

[0121] virtPPP=2virtPPP=1.26×F1 -1.134 =0.857514.

[0122] For control purposes, a PPP reference value of PPP-Ref=0.8869 was determined for the PPP of the same blood sample to be examined in a manner known from the prior art. The virtual reference value thus represents a good estimate of the PPP reference value. Figure 5 The ratio of the measured transmission value to the virtual reference value virtREF and to the measured reference value PPP-Ref is shown over time. Due to the good agreement of the values virtREF and PPP-Ref, Figure 5 The two measurement curves shown in are almost identical.

[0123] In the manner described above, using the mathematical relationships determined in accordance with the database according to the present invention, a virtual reference value (referred to as virt.PPP in the figures) is determined for each of the multiple blood samples to be examined, and a PPP reference value (referred to as ePPP in the figures) is also measured in a manner known from the prior art, in order to compare the virtual reference value with the measured PPP reference value. To this end, an average value is determined from the measured PPP reference values and the virtual reference value, and a percentage difference between the variables is determined. Figure 6 The difference is shown as a function of the mean value, wherein on the scale described, a mean value of 40,000 corresponds approximately to a PPP reference value of 1. This shows that with the aid of the method according to the invention, a virtual reference value can be calculated for a large portion of the blood examined, which represents a very good estimate of the actually measured PPP reference value. Figure 7As illustrated in , in 90% of the cases the difference lies within a small error interval of approximately + / - 4%. The suitability of the method according to the invention for determining a good estimate of the PPP reference value is thus demonstrated.

[0124] Figure 8 The present invention illustrates a device for determining a virtual reference value for performing an LTA measurement. The device comprises an illumination module 13, which is configured to emit a first light wavelength of 405 nm and a second light wavelength of 625 nm, and light 14 having a predetermined intensity in each case. The device also comprises a sample holder 16, into which a transparent cuvette 15 can be inserted manually or automatically. PRP 17 of the blood sample to be examined is located in the cuvette 15. The sample holder is arranged such that the light 14 impinges on a subsection of the cuvette 15 and passes through the PRP 17 disposed therein. The light 14 that has passed through the PRP 17 then strikes a sensor 18, which detects the light intensity and forwards it to a control module 19. The device also comprises an applicator 22, which is configured to apply a predetermined amount of activator to the PRP 17. The lighting module 13 and the applicator 22 are controlled by a control module 19 to carry out the method according to the present invention. Simultaneously, the corresponding measured transmission values xd1, xd2, xd3, and xd4 are detected by sensors and stored in the control module. The control module 19 also includes a calculation module 20, in which the mathematical relationships according to the present invention are stored. Using the mathematical relationships and the measured transmission values, the calculation module 20 determines a virtual reference value for the LTA measurement.

[0125] Alternatively or additionally, a network interface 21 can also be provided, which transmits the measured values xd1 to xd4 detected by the sensors via a data connection to an external computing module, wherein in this case the external computing module takes over the determination of the virtual reference value.

Claims

1. A method for creating a database for determining virtual reference values of PPP reference values of light transmission aggregometry measurements, the method comprising the following steps: a. Provide PRP of reference blood sample; b. performing a light transmission measurement of the PRP of the reference blood sample using a first light wavelength and a second light wavelength different from the first light wavelength, wherein the first light wavelength is in a range between 300 nm and 500 nm and the second light wavelength is in a range between 500 nm and 800 nm, wherein the first light wavelength differs from the second light wavelength by at least 50 nm; c. providing the PPP of the reference blood sample; d. Performing light transmission measurement on PPP for determining a PPP reference value; e. Assigning the measurement result of step d to the measurement result of step b in the database; f. Repeating steps a to e for a plurality of reference blood samples that each differ from one another pairwise in terms of the type and / or concentration of at least one of their components.

2. The method according to claim 1, wherein the first optical wavelength and / or the second optical wavelength satisfies at least one of the following characteristics: - the first light wavelength differs from the second light wavelength by at least 100 nm, - the first light wavelength is in the range between 345 nm and 465 nm, and - the second light wavelength is in the range between 550 nm and 700 nm.

3. The method according to claim 1 or 2, further comprising the steps of: g. After performing the measurement according to step b, adding a predetermined amount of activator to the reference blood sample PRP; h. repeating the measurement according to step b after adding the activator and before the start of aggregation triggered by the activator; i. Assign the measurement result of step h to the measurement result of step d in the database; j. Repeat steps g to i for multiple reference blood samples.

4. The method according to claim 3, wherein the light transmission measurement according to step h is performed within a time period between 0 and 10 s after adding the activating agent. 5 . The method according to claim 4 , wherein during the light transmission measurement according to step h, a time average value of the light transmission is formed over a period of between 1 s and 6 s.

6. The method according to claim 1 or 2, wherein the light transmission measurement of step b is performed using at least three light wavelengths different from each other.

7. The method according to claim 1 or 2, wherein the plurality of reference blood samples comprises a first reference blood sample and a second reference blood sample, wherein the first reference blood sample has at least one first component that is not contained in the second reference blood sample or is contained in the second reference blood sample at a concentration that is less than 1.5 of the concentration of the first component in the first reference blood sample. The method according to claim 7 , wherein the first component is added manually to the first reference blood sample.

9. The method of claim 2, wherein the first light wavelength differs from the second light wavelength by at least 200 nm.

10. The method of claim 2, wherein the first light wavelength is within a range between 385 nm and 425 nm. The method of claim 2 , wherein the second light wavelength is in the range between 600 nm and 640 nm.

12. The method according to claim 4, wherein the light transmission measurement according to step h is performed within a time period between 0 and 5 s after adding the activating agent.

13. The method according to claim 5, wherein during the light transmission measurement according to step h, a time average of the light transmission is formed over a period of between 3 s and 5 s.

14. The method according to claim 7, wherein the first component is contained in the second reference blood sample at a concentration that is less than one-third of the concentration of the first component in the first reference blood sample.

15. The method according to claim 7, wherein the first component is contained in the second reference blood sample at a concentration that is less than one-fifth of the concentration of the first component in the first reference blood sample.

16. The method of claim 7, wherein the first component is selected from the group consisting of hemoglobin, ceruloplasmin, lipoprotein, triglyceride, and bilirubin.

17. A method for determining a virtual reference value for a PPP reference value of a light transmission aggregometry measurement of PRP of a blood sample to be examined using a database created by the method according to any one of claims 1 to 16, the method comprising the following steps: a. providing the PRP of the blood sample to be examined; b. performing light transmission measurement of the PRP of the blood sample to be examined using a first light wavelength and a second light wavelength different from the first light wavelength; c. Using the measurement results obtained in step b and the database, a virtual reference value of the blood sample to be examined is determined.

18. The method according to claim 17, further comprising the steps of: d. After performing the measurement according to step b, adding a predetermined amount of activator to the PRP of the blood sample to be examined; e. After adding the activator to the PRP of the blood sample to be examined and before the aggregation is initiated by the activator, the measurement according to step b is repeated; f. Incorporating the measurement results obtained in step e into the determination in step c.

19. The method according to claim 17, wherein a mathematical relationship is generated based on the database, which has measurement results obtained on the blood sample to be examined as input variables and a virtual reference value of the blood sample to be examined as output variable.

20. A method for performing light transmission aggregometry measurements on a blood sample to be examined, the method comprising the steps of: a. Execution of the method according to any one of claims 17 to 19 for determining a virtual reference value of the blood sample to be examined, b. Performing a light transmission aggregometry measurement using the virtual reference value.

21. A device for determining a virtual reference value for performing LTA measurement on PRP of a blood sample to be examined, the device comprising an illumination module (13) for selectively emitting light of a first light wavelength and a second light wavelength different from the first light wavelength; a sample receiving portion (16) for inserting a PRP sample (17) so that the light of the illumination module (13) passes through the PRP (17); a light sensor (18) configured to detect the light passing through the PRP (17); and a control module (19) configured to operate the device so that the following steps are performed: a. performing a light transmission measurement of the PRP of the blood sample to be examined using a first light wavelength and a second light wavelength different from the first light wavelength, wherein the first light wavelength is in the range between 300 nm and 500 nm and the second light wavelength is in the range between 500 nm and 800 nm, wherein the first light wavelength differs from the second light wavelength by at least 50 nm; b. Determine a virtual reference value for the blood sample to be examined using the measurement results obtained in step a and the database created according to the method of any one of claims 1 to 16.

22. The device according to claim 21, comprising an applicator (22) for automatically adding a predetermined amount of activating agent to the PRP (17), wherein the control module (19) is configured to control the device so as to perform the following steps: c. After performing the measurement according to step a, adding a predetermined amount of activator to the PRP of the blood sample to be examined; d. after adding the activator to the PRP of the blood sample to be examined and before the aggregation triggered by the activator is started, repeating the measurement according to step a; e. Incorporate the measurement results obtained in step d into the measurement in step b.

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