METHOD FOR CREATING A DATABASE FOR DETERMINING A LIGHT TRANSMISSION AGGREGOMETRY REFERENCE VALUE, AND METHOD AND DEVICE FOR CARRYING OUT A LIGHT TRANSMISSION AGGREGOMETRY MEASUREMENT

Abstract

What is disclosed is a method for creating a database for determining a virtual reference value for a light transmission aggregometry measurement, comprising the following steps: a. providing platelet-rich plasma PRP (17) of a reference blood sample; b. performing a light transmission measurement with a first light wavelength and a second light wavelength (14), different from the first, on the PRP (17) of the reference blood sample; c. providing platelet-poor plasma PPP of the reference blood sample; d. performing a light transmission measurement on the PPP in order to determine a PPP reference value; e. assigning the measurement result of step d to the measurement results of step b in a database; f. repeating steps a to e for a plurality of reference blood samples.

The database obtained using this method makes it possible to determine a virtual reference value that is an excellent estimate of the PPP reference value. Once the database has been created, it is possible to determine virtual reference values of blood samples to be examined and perform LTA measurements using the virtual reference values, without the need to obtain PPP from the blood samples to be examined.

Claims

1. A method for creating a database for determining a virtual reference value for a PPP reference value of a light transmission aggregometry measurement, comprising the following steps: a) providing platelet-rich plasma (PRP) of a reference blood sample; b) performing a light transmission measurement with a first light wavelength and a second light wavelength, different from the first, on the PRP of the reference blood sample, wherein the first wavelength is in a range between 300 nm and 500 nm and the second 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 platelet-poor plasma (PPP) of the reference blood sample; d) performing a light transmission measurement on the PPP in order to determine a PPP reference value; e) assigning the measurement result of step d to the measurement results of step b in a database; f) repeating steps a to e for a plurality of reference blood samples that each differ from one another in pairs in terms of the type and/or concentration of at least one of their substances.

2. The method of claim 1, wherein the first and/or second wavelength satisfy at least one of the following features: the first wavelength differs from the second wavelength by at least 100 nm, preferably by at least 200 nm, the first wavelength is in a range between 345 nm and 465 nm, more preferably between 385 nm and 425 nm, and the second wavelength is in a range between 550 nm and 700 nm, more preferably between 600 nm and 640 nm.

3. The method of claim 1, comprising the following further steps: g) adding a predefined amount of an activator to the PRP of the reference blood sample after performing the measurement according to step b; h) repeating the measurement according to step b after adding the activator and before introducing an aggregation triggered by the activator; i) assigning the measurement result of step h to the measurement result of step d in the database; j) repeating steps g to i for the plurality of reference blood samples.

4. The method of claim 3, wherein the light transmission measurement according to step h is performed in a time period between 0 and 10 s, preferably between 0 and 5 s after the addition of the activator.

5. The method of claim 4, wherein, during the light transmission measurement according to step h, a temporal average value of the light transmission is formed over a time period between 1 s and 6 s, preferably between 3 s and 5 s.

6. The method of claim 1, wherein the light transmission measurements of step b are performed with at least three mutually different light wavelengths.

7. The method of claim 1, 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 contains at least one first substance that is not contained in the second reference blood sample or is contained in the second reference blood sample in a concentration that is less by a factor of more than 1.5, preferably by a factor of more than 3, more preferably by a factor of more than 5 than the concentration of the first substance in the second reference blood sample, wherein the first substance is preferably selected from the group consisting of hemoglobin, ceruloplasmin, lipoprotein, triglycerides, bilirubin.

8. The method of claim 7, wherein the first substance of the first reference blood sample is added manually.

9. A method for determining a virtual reference value for a PPP reference value of a light transmission aggregometry measurement on the PRP of a blood sample to be examined using a database of claim 1, comprising the following steps: a) providing PRP of the blood sample to be examined; b) performing a light transmission measurement with a first light wavelength and a second light wavelength, different from the first, on the PRP of the blood sample to be examined; c) using the measurement results obtained by step b and the database to determine the virtual reference value of the blood sample to be examined.

10. The method of claim 9, furthermore comprising the following steps: d) adding a predefined amount of an activator to the PRP of the blood sample to be examined after performing the measurement according to step b; e) repeating the measurement according to step b after adding the activator to the PRP of the blood sample to be examined and before introducing an aggregation triggered by the activator; f) incorporating the measurement results obtained by step e into the determination of step c.

11. The method of claim 9, wherein a mathematical relationship based on the database is established, this relationship having the measurement results obtained on the blood sample to be examined as input variables and the virtual reference value of the blood sample to be examined as output variable.

12. A method for performing a light transmission aggregometry measurement on a blood sample to be examined, comprising the following steps: a) performing the method of claim 9 for determining the virtual reference value of the blood sample to be examined, b) performing a light transmission aggregometry measurement using the virtual reference value.

13. A device for determining a virtual reference value for performing an LTA measurement on the PRP of a blood sample to be examined, comprising an illumination module (13) for selectively emitting light of a first light wavelength and of a second light wavelength different from the first, a sample receptacle (16) for the introduction of a PRP sample (17) such that the light from the illumination module (13) passes through the PRP (17), a light sensor (18) that is designed to capture the light that has passed through the PRP (17), and a control module (19) that is designed to drive the device such that the following steps are carried out: a) performing a light transmission measurement with a first light wavelength and a second light wavelength, different from the first, on the PRP of the blood sample to be examined, wherein the first wavelength is in a range between 300 nm and 500 nm and the second 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; b) using the measurement results obtained by step a and a database of claim 1 to determine a virtual reference value of the blood sample to be examined.

14. The device of claim 13, having an applicator (22) for automatically adding a predefined amount of an activator to the PRP (17), wherein the control module (19) is designed to drive the device such that the following steps are carried out: c) adding a predefined amount of an activator to the PRP of the blood sample to be examined after performing the measurement according to step a; d) repeating the measurement according to step a after adding the activator to the PRP of the blood sample to be examined and before introducing an aggregation triggered by the activator; e) incorporating the measurement results obtained by step d into the determination of step b.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Preferred exemplary embodiments of the invention are explained in detail below with reference to the accompanying drawings, in which:

[0068] FIG. 1: shows examples of light transmission measurements that were performed on the PRP of three reference blood samples with a first wavelength;

[0069] FIG. 2: shows the light transmission measurements from FIG. 1, wherein the measurement curves were shifted to the zero point at the time t=?8 s;

[0070] FIG. 3: shows the ratio between the transmittances shown in FIG. 2 and the respective PPP reference value of the respective reference blood sample over time;

[0071] FIG. 4: shows one example of an LTA measurement that was performed on the PRP of a blood sample to be examined;

[0072] FIG. 5: shows the ratio between the transmittance shown in FIG. 4 and the virtual reference value and also the measured PPP reference value of the blood sample to be examined over time;

[0073] FIG. 6: shows a comparison between measured PPP reference values and virtual reference values for a large number of blood samples to be examined, as determined using the method according to the invention;

[0074] FIG. 7: shows a statistical evaluation of the results shown in FIG. 6;

[0075] FIG. 8: shows a schematic view of a device according to the invention for determining a virtual reference value.

DETAILED DESCRIPTION

[0076] FIG. 1 shows three exemplary light transmission measurements over time that were performed on the PRP of three different reference blood samples. The change in the measured value of an intensity meter is plotted as a function of time. The measured value is proportional to the common logarithm of the transmittance (the transmittance is referred to below using the letter T). An increase in the measured value thus corresponds to an increase in the transmittance. The measurements span the time period t=?8 s to t=6 min, wherein, at the time t=0 s, the activator adenosine diphosphate (ADP) was added at a volume ratio of 1:9 to the respective PRP sample (one part ADP, 9 parts PRP). The measurements were performed with a wavelength of ?1=625 nm. Reference blood samples 1 to 3 differ from one another only in the type of substance added manually to the respective sample. During reference blood sample 1, no substance was added manually, intralipids were added to reference blood samples 2 and 3 at concentrations of 75 mg/dl and 151 mg/dl, respectively. It may be seen in FIG. 1 that the addition of the intralipids leads to a lower absolute value of the transmittance.

[0077] FIG. 2 shows the measurements from FIG. 1, wherein the curves have been shifted to the zero point in order to make the changes in the transmittance clearer. It may be seen that the transmittance of reference sample 1 hardly changes, or even decreases, shortly after the addition of the activator (between t=0 and t=10 s). During this time period, the platelet activation process begins without any aggregation already having taken place. After the end of the time period, the platelet cross-linking begins at around t=10 s, and aggregates form in the PRP, as a result of which the transmittance increases over time. After around t=150 s, a maximum transmittance is reached, which then hardly changes.

[0078] For reference samples 2 and 3, on the other hand, it is possible to see a sudden change by ?(log.sub.10 T)=0.03 and by ? (log.sub.10 T)=0.05, respectively, at the time t=0. For these reference blood samples too, the aggregation then begins (from around t=10 s) and the transmittance continues to increase.

[0079] The absolute value of the change in the transmittance has no significance per se, since it depends for example on the concentration of platelets contained in the sample and on the concentration and type of other substances contained therein. For this reason, in the prior art, it is necessary to determine a respective PPP reference value through a light transmission measurement on the PPP of the same blood sample and to correlate the measured values obtained on the PRP with this measured PPP reference value. Such PPP reference values were determined, in the reference blood samples shown above, in a manner known from the prior art. FIG. 3 shows the corresponding ratio of the transmittances of reference blood samples 1 to 3 to the respective PPP reference value overtime. It may be seen that the ratio over time approaches a value of around 85 for all three samples. From the measured data shown in FIG. 3, it is possible to draw conclusions about the functionality of the platelets.

[0080] The method according to the invention for creating a database is explained below by way of example with reference to the measured data shown in FIGS. 1 to 3. In addition to the transmission measurements shown in FIGS. 1 to 3, a respective light transmission measurement with the wavelength ?2=405 nm was performed on the PRP of the same reference blood samples shortly before the addition of the activator (at t=?3 s) and shortly after the addition of the activator (by forming an average value in the time period between t=2 s and 6 s). By way of example, the following measurement results were obtained for reference blood sample 1: [0081] the transmittance at the wavelength ?1 before the addition of the activator at the time t=?3 s: xd1=0.82676788; [0082] the transmittance at the wavelength ?2 before the addition of the activator at the time t=?3 s: xd2=0.532220558; [0083] the transmittance at the wavelength ?1 after the addition of the activator, obtained by averaging in the time period between t=2 s and t=6 s: xd3=0.840990463; [0084] the transmittance at the wavelength ?2 after the addition of the activator, obtained by averaging in the time period between t=2 s and t=6 s: xd4=0.551279411, and [0085] the PPP reference value measured on the PPP of the reference blood sample: PPP-Ref=1.0228.

[0086] The values xd1, xd2, xd3, xd4 and the PPP reference value were added to a database. Corresponding values were also determined for reference blood samples 2 and 3 and were likewise added to the database. The database is illustrated in Table 1 below.

TABLE-US-00001 TABLE 1 Sample no. 1 2 3 added intralipid intralipid substance 75 mg/dl 151 mg/dl xd1 0.82676788 0.362703212 0.166929278 xd2 0.532220558 0.092530236 0.027191926 xd3 0.840990463 0.38363385 0.188179259 xd4 0.551279411 0.102101591 0.027871212 PPP-Ref 1.0228 0.4433 0.2118 virtREF 1.0409 0.460 0.205

[0087] Corresponding measured values were captured for further reference blood samples 4 to 7 and likewise added to the database. Reference blood samples 4 to 7 differed from one another only in terms of the respective substances added manually to the PRP. The substances and the concentration thereof are listed, together with the captured measured values, in Table 2 below.

TABLE-US-00002 TABLE 2 sample no. 4 5 6 7 added 15 mg/dl 568 mg/dl 372 mg/dl substance bilirubin hemoglobin triglycerides xd1 0.91048 0.881060 0.852417 0.557092 xd2 0.56510 0.017670 0.129883 0.214720 xd3 0.92432 0.88447 0.862035 0.593829 xd4 0.58818 0.018583 0.151792 0.236192 PPP-Ref 1.0184 1.0160 0.9520 0.6849 virtREF 1.01991 1.01034 0.9831 0.69194

[0088] Within the database, it is possible to recognize patterns that are based on the influence of the additionally added substances. Comparing the transmission values reveals for example that the addition of a substance may have a specific influence both on the determined transmission values and on the measured PPP reference values. By way of example, a comparison of the values xd1 and xd2 of samples 4 to 6 shows that a change in transmittance triggered by the addition of bilirubin or hemoglobin is significantly more pronounced at the wavelength ?2 than at the wavelength ?1.

[0089] Another recognizable pattern is for example that, for sample 1, the addition of the activator causes only a small change between the transmission values xd1 and xd3, while the change for samples 2 and 3 (that is to say with an increase in intralipid concentration) increases significantly (see FIG. 2). At the same time, the addition of the substances leads to a more or less pronounced change in the PPP reference value.

[0090] After the measurements explained above by way of example have been performed on a large number of reference blood samples and recorded in the database, the type of patterns described above leads to statistically significant relationships that make it possible to establish a mathematical relationship in order to determine a virtual reference variable.

[0091] One example of a mathematical relationship is explained below:

The variables:

[00001]$V1=\mathrm{xd}1/\mathrm{xd}2,V2=\mathrm{xd}3/\mathrm{xd}4,V3=V2/V1,\mathrm{Ve}=\left(V1+V2\right)/e^{V3}\mathrm{and}{X}^{\mathrm{xdPIP}}=\mathrm{xd}4+\mathrm{xd}3-\mathrm{xd}2-\mathrm{xd}1$

are determined. In addition, the variable

[00002]$X^{\mathrm{vPIP}}=\left(1-\mathrm{xd}3\right)?\left(-{\log }_{10}\left(\mathrm{xd}3\right)\right)?\left(V1+\mathrm{Ve}\right)/e^{\mathrm{Ve}}$

is determined. Based on the variables referenced above, a preliminary virtual reference variable 1virtPPP may be determined as follows:

[00003]$1\mathrm{virtPPP}=1+X^{\mathrm{xdPIP}}-X^{\mathrm{vPIP}}.$

In addition, the factor

[00004]$F1=\mathrm{xd}3/1\mathrm{virtPPP}$

is calculated. It turned out that, for those PRP samples in which the value of the factor F1 is less than a threshold value (in the present example F1<1.19), the preliminary virtual reference variable 1virtPPP is a good estimate for the PPP reference value.

[0092] If the factor F1 is greater than the threshold value (in the present example, that is to say F1>1.19), then a corrected virtual reference value 2virtPPP is determined as follows:

[00005]$2\mathrm{virtPPP}=1.26?F{1}^{-1.134}.$

[0093] For those PRP samples in which the factor is F1>1.19, the corrected virtual reference value 2virtPPP is a good estimate for the PPP reference value. The mathematical relationship, reproduced below, for ascertaining the virtual reference value virtPPP was thus determined from the database in this case:

[00006]$\mathrm{virtPPP}=\{\begin{array}{cc}1\mathrm{virtPPP}=1+X^{\mathrm{xdPIP}}-X^{\mathrm{vPIP}},& \mathrm{if}F1<1.19\\ 2\mathrm{virtPPP}=1.26?F{1}^{-1.134},& \mathrm{if}F1>1.19\end{array}$

[0094] The virtual reference values virtPPP thereby determined for reference samples 1 to 6 are indicated in Tables 1 and 2 above. The tables show that there is a good match with the measured PPP reference values (PPP-Ref).

[0095] FIG. 4 shows an LTA measurement on the PRP of a blood sample to be examined, which was performed under the same measurement conditions as the measurements shown in FIGS. 1 and 2 in a time period between t=?8 s and t=6 min, wherein the measurement curve is shifted to the zero point at the time t=?8 s. At the time t=0 s, the activator ADP was added to the PRP in a ratio of 9:1. The following transmission values xd1, xd2, xd3, xd4 were ascertained for the wavelengths ?1=625 nm, ?2=405 nm in the manner already described above before and after the addition of the activator, respectively:

[00007]$\mathrm{xd}1=0.698934,\mathrm{xd}2=0.257505,\mathrm{xd}3=0.726952,\mathrm{xd}4=0.280035.$

[0096] The mathematical relationship described above was used to calculate the abovementioned intermediate values as follows:

TABLE-US-00003 V1 V2 V3 X.sup.xdPIP V.sup.e X.sup.vPIP 2.714 2.596 0.956 0.05055 2.04055443 0.0264134

[0097] The provisional virtual reference value was:

[00008]$1\mathrm{virtPPP}=1.0259$

[0098] In addition, the factor was determined.

[00009]$F1=1.411.$

[0099] Since the factor exceeds the value of 1.19, the resulting virtual reference value was:

[00010]$\mathrm{virtPPP}=2\mathrm{virtPPP}=1.26?F{1}^{-1.134}=0.857514.$

[0100] For control purposes, a PPP reference value PPP-Ref=0.8869 was ascertained on the PPP of the same blood sample to be examined in a manner known from the prior art. The virtual reference value is thus a good estimate for the PPP reference value. FIG. 5 shows the ratio of the measured transmission value to the virtual reference value virtREF and to the measured reference value PPP-Ref over time. Owing to the good match between the values virtREF and PPP-Ref, the two measurement curves shown in FIG. 5 are almost identical.

[0101] In the manner described above and using the mathematical relationship determined based on the database according to the invention, a respective virtual reference value (referred to as virt.ppp in the figure) was determined for a plurality of blood samples to be examined and, in addition, a PPP reference value (referred to as ePPP in the figure) was measured in a manner known from the prior art in order to compare the virtual reference value in each case with the measured PPP reference value. For this purpose, the average value of the measured PPP reference value and the virtual reference value was determined and the percentage difference between the variables was ascertained. FIG. 6 shows this difference as a function of the average value, wherein, in the scale shown, an average value of 40000 corresponds approximately to a PPP reference value of 1. It turns out that, using the method according to the invention, it was possible to calculate a virtual reference value for a majority of the examined blood samples, which reference value represents a very good estimate for the PPP reference value actually measured. As illustrated in FIG. 7, the difference, in 90% of cases, is within a small error interval of around +/?4%. The suitability of the method according to the invention for ascertaining a good estimate for the PPP reference value was thus confirmed.

[0102] FIG. 8 shows a device according to the invention for determining a virtual reference value for performing an LTA measurement. The device comprises an illumination module 13, which is designed to emit light beams 14 of a first light wavelength of 405 nm and of a second light wavelength of 625 nm and with a predefined intensity in each case. The device furthermore comprises a sample holder 16 into which a transparent cuvette 15 is able to be introduced manually or else automatically. The cuvette 15 contains PRP 17 of a blood sample to be examined. The sample holder is arranged such that the light beams 14 impinge on a subregion of the cuvette 15 and pass through the PRP 17 arranged therein. The light beams 14 that have passed through the PRP 17 then impinge on a sensor 18, which captures the intensity of the light beams and forwards same to a control module 19. The device furthermore has an applicator 22 that is designed to add a predefined amount of an activator to the PRP 17. The illumination module 13 and the applicator 22 are controlled by the control module 19 so as to perform the method according to the invention, wherein the respective measured transmission values xd1, xd2, xd3 and xd4 are simultaneously captured by the sensor and stored in the control module. The control module 19 furthermore comprises a computing module 20 in which the mathematical relationship according to the invention is stored. Using the mathematical relationship and the measured transmission values, the computing module 20 determines a virtual reference value for the LTA measurement.

[0103] As an alternative or in addition, provision may also be made for a network interface 21 that transmits the measured values xd1 to xd4 captured by the sensor to an external computing module via a data connection, wherein the determination of a virtual reference value is in this case taken over by the external computing module.