GLOBAL TEST FOR DETERMINING THE STATUS OF THE BLOOD COAGULATION SYSTEM

Abstract

Embodiments of the present invention are in the field of blood coagulation diagnostics and relate to a test for establishing an individual's blood coagulation system status based on the amount of in-vitro-generated F1+2 peptide, and to a test kit for use in such a method.

Claims

1. A method for establishing an individual's blood coagulation system status, the method comprising: providing a reaction mixture by adding a coagulation activator to a sample from the individual; incubating the reaction mixture; quantitatively determining an amount of F1+2 in the reaction mixture; and establishing the blood coagulation system status by comparing the amount of F1+2 in the reaction mixture with a reference value or reference value range that represents a normal blood coagulation system status of a healthy individual.

2. The method as claimed in claim 1, wherein a procoagulatory status of an individual's blood coagulation system is established when the amount of F1+2 quantitatively determined is above the reference value or reference value range that represents a normal blood coagulation system status of a healthy individual.

3. The method as claimed in claim 1, wherein an anticoagulatory status of an individual's blood coagulation system is established when the amount of F1+2 quantitatively determined is below the reference value or reference value range that represents a normal blood coagulation system status of a healthy individual.

4. The method as claimed in claim 1, wherein the reaction mixture is diluted after the incubating the reaction mixture and the quantitative determination of the amount of F1+2 is carried out in the reaction mixture thus diluted.

5. The method as claimed in claim 4, wherein the quantitative determination of the amount of F1+2 in the reaction mixture or in the diluted reaction mixture in is carried out using a F1+2-specific immunoassay.

6. The method as claimed in claim 5, wherein the F1+2-specific immunoassay comprises use of a first antibody having specificity for F1+2 and use of a second, F1+2-binding antibody or of a second antibody having specificity for the immune complex consisting of F1+2 and the first antibody.

7. The method as claimed in claim 6, wherein the first and second antibodies are each associated with a particulate solid phase and the quantitative determination of the amount of F1+2 comprises: mixing the reaction mixture with the particulate solid phases associated with the first and second antibodies; and measuring an agglutination of the particulate solid phases in the reaction mixture.

8. The method as claimed in claim 7, wherein the agglutination of the particulate solid phases in the reaction mixture is measured photometrically.

9. The method as claimed in claim 6, wherein the first antibody is associated with a first particulate solid phase and the second antibody is associated with a second particulate solid phase, and wherein the first particulate solid phase is associated with a first component of a signal-forming system and the second particulate solid phase is associated with a second component of a signal-forming system, and wherein the first and the second component of the signal-forming system interact with one another such that this produces a detectable signal when the first and the second component of the signal-forming system are brought into spatial proximity and an agglutination of the particulate solid phases in the reaction mixture is measured based on of the signal produced.

10. The method as claimed in claim 9, wherein the first component of the signal-forming system is a chemiluminescent agent and the second component of the signal-forming system is a photosensitizer or the first component of the signal-forming system is the photosensitizer and the second component of the signal-forming system is the chemiluminescent agent, and wherein a chemiluminescence in the reaction mixture is measured.

11. The method as claimed in claim 1, wherein the reaction mixture is incubated for a period of from 5 seconds to 60 minutes.

12. The method of claim 1, wherein the reaction mixture is incubated for a period of from 5 to 20 minutes.

13. The method as claimed in claim 1, wherein the coagulation activator is an activator of the plasmatic coagulation system.

14. The method as claimed in claim 13, wherein the activator of the plasmatic coagulation system is selected from the group of thromboplastin, factor IIa, factor VIIa, factor IXa, factor Xa, factor XIa, factor XIIa, snake venoms, negatively charged phospholipids, calcium ions, tissue factor, silica, kaolin, ellagic acid, Celite, and polyphosphates.

15. The method as claimed in claim 1, wherein the coagulation activator is a platelet activator.

16. The method as claimed in claim 15, wherein the platelet activator is selected from the group of ADP, epinephrine, collagen, thrombin-receptor-activating peptide, thromboxane A2 mimic U46619, arachidonic acid, ristocetin, thrombin, von Willebrand factor, collagen-related peptide, convulxin, calcium ionophore A23187, and phorbol 12-myristate 13-acetate.

17. The method as claimed in claim 1, wherein the coagulation activator is a chemical or physical factor that heightens the procoagulatory effect of erythrocytes.

18. The method as claimed in claim 1, wherein the coagulation activator is a leukocyte-activating factor.

19. A test kit for establishing a blood coagulation system status of an individual, the test kit comprising: a reagent comprising a coagulation activator; and one or more reagents for the quantitative determination of F1+2.

20. The test kit as claimed in claim 19, further comprising: a first calibrator material that represents a normal blood coagulation system status of a healthy individual; and optionally, at least a second calibrator material that represents a procoagulatory or an anticoagulatory blood coagulation system status of an individual.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0070] FIG. 1 shows a diagram in which the linear relationship between the measured amount of F1+2 [kcnt] and dilution (undiluted, 1:2 and 1:4) in the various sample types (whole blood, platelet-rich plasma (PRP), platelet-poor plasma (PPP)) is made clear.

[0071] FIG. 2 shows a diagram depicting the calibration curve for the inventive test for determining the amount of in-vitro-generated F1+2.

[0072] FIG. 3 shows a diagram in which F1+2 generation determined according to an embodiment of the present invention (% of norm) is compared with the ETP (parameter=Cmax; % of norm) in various samples.

[0073] FIG. 4 shows a diagram in which F1+2 generation determined according to an embodiment of the present invention (% of norm) is compared with the ETP (parameter=AUC; % of norm) in various samples.

[0074] FIG. 5 shows four diagrams in which F1+2 generation determined according to an embodiment of the present invention and the endogenous thrombin potential (ETP) of plasma samples deficient in different coagulation factors (factors II, VII, IX, and X) are compared.

[0075] FIG. 6 shows a diagram in which F1+2 generation potential determined according to an embodiment of the present invention with varying incubation times and the amount of rivaroxaban determined with an anti-FXa test are compared in plasma samples having different rivaroxaban concentrations.

[0076] FIG. 7 shows a diagram in which F1+2 generation potential determined according to an embodiment of the present invention and the amount of dabigatran determined with an anti-IIa test are compared in plasma samples having different dabigatran concentrations.

[0077] FIG. 8 shows a diagram depicting the F1+2 generation potential determined according to an embodiment of the present invention in a plasma sample containing 120 ng/mL of dabigatran with different incubation times.

[0078] FIG. 9 shows two diagrams in which F1+2 generation determined according to an embodiment of the present invention (% of norm) employing just a 5-minute incubation time after addition of all coagulation factors is compared with the ETP (parameter=Cmax; % of norm, left diagram; parameter=AUC; % of norm, right diagram) in various samples.

[0079] FIG. 10 shows diagrams depicting F1+2 generation determined according to an embodiment of the present invention (kcnt) in collagen-preactivated samples of whole blood (WB), PRP, and PPP from three blood donors.

[0080] FIG. 11 shows a diagram depicting F1+2 generation determined according to an embodiment of the present invention (kcnt) in collagen-preactivated plasma samples having different platelet counts.

[0081] FIG. 12 shows a diagram in which a lupus anticoagulant coagulation test is compared with the lupus anticoagulant F1+2 generation test of an embodiment of the present invention. In the “original” test run, the sample is used as is, and in the “mixture” test run the sample is diluted 1:1 with a normal plasma.

[0082] FIG. 13 shows a diagram depicting F1+2 generation determined according to an embodiment of the present invention (kcnt) in whole blood (WB), PRP, and PPP with/without inhibition of platelets by prostaglandin E1 (PGE1) and with/without subsequent activation of platelets by collagen.

DETAILED DESCRIPTION

Examples

Example 1: Determination of the Amount of In-Vitro-Generated F1+2 Peptide in Plasma Samples and Whole Blood Samples

[0083] Samples of whole blood or plasma (PRP or PPP) were mixed with the components of the Innovance ETP assay (Siemens Healthcare Diagnostics Products GmbH, Germany) as follows: [0084] 87 μL ETP buffer [0085] +40 μL ETP reagent [0086] 265 seconds incubation [0087] +108 μL sample [0088] 440 seconds incubation [0089] +15 μL Innovin reagent (lipidated tissue factor; Siemens Healthcare Diagnostics Products GmbH, Germany) [0090] +10 μL ETP CaCl.sub.2) solution [0091] 20 minutes incubation at 37° C. [0092] 1:100 dilution of the reaction mixture by addition of Owren's Veronal Buffer (OVB buffer). [0093] 5 μL of the diluted reaction mixture was mixed with [0094] a first reagent (“reagent BA”) comprising a first, biotinylated monoclonal antibody having specificity for the neoepitope of F1+2; and then [0095] with a second reagent (“Chemibeads reagent”) comprising a second monoclonal antibody having specificity for the immune complex consisting of the first antibody and F1+2 peptide bound thereto, which is coupled to latex particles associated with a chemiluminescent compound (Chemibeads); and [0096] with a third reagent (“Sensibeads” reagent) comprising streptavidin-coated latex particles associated with a photosensitizer (Sensibeads).

[0097] After further incubation of the reaction run for a period of 6 minutes, the chemiluminescence signal in the arbitrary unit kilocounts (kcnt) was measured.

[0098] The LOCI® technology used here is based on a chemiluminescent compound coupled to latex particles (Chemibeads, CB) and a photosensitizer coupled to latex particles (Sensibeads, SB) being brought into spatial proximity by binding to an analyte (here: F1+2 peptide), with the result that singlet oxygen generated by the photosensitizer is able to excite the chemiluminescent compound. The amount of chemiluminescence generated correlates with the amount of analyte.

[0099] This was used to investigate samples from three apparently healthy normal donors. Each sample underwent a duplicate determination and all results were averaged (Table 1). The results show that, in the presence of tissue factor, lipids, and CaCl.sub.2) from the Innovance ETP reagents and without addition of a platelet activator, the F1+2 generation potential in PRP and PPP is almost equally high. Since the hematocrit of 36 to 53% (normal range in men and women) means that the actual plasma volume in whole blood available for the measurement of F1+2 is only 47 to 64%, the value of 1242 kcnt determined in whole blood is very plausible when set alongside the value of 2111 kcnt determined in plasma. At the same time, this demonstrates that the high dilution ensures that the turbidity caused by the high platelet count in PRP and by the high total cell count in whole blood does not substantially interfere with the F1+2 determination.

TABLE-US-00001 TABLE 1 Sample F1 + 2 amount [kcnt] Whole blood 1242 PRP 2009 PPP 2111

[0100] To investigate the dependence of the amount of in-vitro-generated F1+2 on the available coagulation factors, the samples were prediluted 1:2 and 1:4 with OVB buffer (Table 2).

TABLE-US-00002 TABLE 2 Mean F1 + 2 amount [kcnt] value SD CV Donor Sample Dilution 1 2 [kcnt] [kcnt] (%) 1 Whole 1:1 1367 1517 1442 106.0 7.3 blood 1:2 622 615 618 4.9 0.8 1:4 298 291 294 5.1 1.7 2 1:1 1213 1278 1246 45.8 3.7 1:2 538 473 505 45.8 9.1 1:4 231 217 224 10.1 4.5 3 1:1 1041 1033 1037 5.8 0.6 1:2 481 485 483 3.2 0.7 1:4 218 215 217 2.0 0.9 1 PRP 1:1 2379 2498 2439 84.2 3.5 1:2 1172 1083 1127 62.9 5.6 1:4 521 561 541 28.4 5.2 2 1:1 1984 1901 1942 58.7 3.0 1:2 957 895 926 44.2 4.8 1:4 451 407 429 31.0 7.2 3 1:1 1689 1603 1646 60.8 3.7 1:2 728 774 751 32.0 4.3 1:4 372 356 364 11.2 3.1 1 PPP 1:1 2705 2478 2592 160.9 6.2 1:2 1404 1215 1309 133.3 10.2 1:4 592 574 583 13.2 2.3 2 1:1 2044 1888 1966 110.5 5.6 1:2 951 942 947 5.9 0.6 1:4 451 423 437 19.7 4.5 3 1:1 1865 1687 1776 125.8 7.1 1:2 884 822 853 44.3 5.2 1:4 404 379 391 17.3 4.4

[0101] In all three sample types, a linear relationship was found between the content in the sample (i.e. content of coagulation factors) and the amount of in-vitro-generated F1+2. This confirms the dilution linearity of the test results and, in the case of whole blood and PRP as sample, hence also the absence of potential interferences brought about solely by the presence of cells (for example the “inner filter effect”) and by the turbidity of the sample (FIG. 1).

Example 2: Comparison of the Inventive F1+2 Generation Assay with a Thrombin Generation Assay (Endogenous Thrombin Potential

[0102] For the performance of the inventive method for determining the amount of in-vitro-generated F1+2, plasma samples were mixed with the components of the Innovance ETP assay (Siemens Healthcare Diagnostics Products GmbH, Germany) as follows: [0103] 108 μL sample [0104] +82 μL ETP buffer [0105] +35 μL ETP reagent [0106] 150 seconds incubation at 37° C. [0107] +15 μL Innovin reagent (lipidated tissue factor) [0108] +10 μL ETP CaCl.sub.2 solution [0109] 20 minutes incubation at 37° C. [0110] 1:100 dilution of the reaction mixture by addition of OVB buffer.

[0111] As in example 1, 5 μL of the diluted reaction mixture was used for determining the amount of F1+2 formed in the reaction mixture.

[0112] A calibration of the inventive F1+2 generation assay using a plasma calibrator material having a defined prothrombin content (% of norm) (Innovance ETP standard, Siemens Healthcare Diagnostics Products GmbH, Germany) was additionally carried out. For the determination of the calibration curve (FIG. 2), the standard was diluted with OVB buffer. The highest calibration point at 196% was not measured, but extrapolated.

[0113] For comparison with the endogenous thrombin potential (ETP), the plasma samples were additionally measured with the Innovance ETP assay (Siemens Healthcare Diagnostics Products GmbH, Germany). In this known method for determining the ETP, a coagulation activator (Innovin and calcium ions), a chromogenic thrombin substrate, and a fibrin inhibitor are added to the sample and the reaction kinetics of a thrombin substrate are determined photometrically over a period of about 20 minutes. The ETP is determined on the basis of the parameters Cmax (maximum rate of reaction) and AUC (area under the curve) of the measured reaction kinetics. A calibration curve is created using the Innovance ETP standard (Siemens Healthcare Diagnostics Products GmbH, Germany), with the results given units of % of norm. The ETP test undergoes automated processing on the BCS XP analysis system (Siemens Healthcare Diagnostics Products GmbH, Germany).

[0114] For the purposes of comparing F1+2 generation and endogenous thrombin potential, plasma samples were produced by diluting a normal plasma pool (mixture of plasmas from a plurality of healthy donors) with a deficient plasma pool (mixture of plasmas from a plurality of healthy donors in which a severe deficiency in a plurality of coagulation factors has been produced by AlOH absorption) in a number of dilution steps. FIGS. 3 and 4 show that, in the ETP test and in the F1+2 generation test the depletion in coagulation factors is manifested in a similar manner in a fall in % of norm values.

[0115] This shows that the F1+2 generation test, the measurement principle and evaluation of which are considerably simpler than the measurement principle and evaluation of the endogenous thrombin potential, responds in a similar manner to the loss of coagulation factors and thus detects an anticoagulatory situation in a similar manner to the thrombin generation test.

Example 3: Comparative Determination of Single-Factor Deficiencies by Measuring the F1+2 Generation Potential and Measuring the Endogenous Thrombin Potential (ETP

[0116] To determine the dependence of the F1+2 generation potential on individual coagulation factors, single-factor deficient samples were produced. This was done by diluting a normal plasma pool with various factor-deficient plasmas (plasmas deficient in factors II, VII, IX, and X) in various dilution steps. These samples were measured both with the Innovance ETP thrombin generation test and with the inventive F1+2 generation test, as per example 2.

[0117] The measured values generally show a fall as a function of the factor concentration in a similar manner both in the thrombin generation test and in the F1+2 generation test (FIG. 5). The dependence of the factor II activity is in both cases very strong and almost linear. A deficiency in factor VII has only a weak effect and becomes noticeable only below 20% of the norm. A deficiency in factor IX did not show any effects at all. Factor X deficiency has an effect only below 1% of the norm, both thrombin generation and F1+2 generation being hindered completely in pure deficient plasma.

[0118] The F1+2 generation test thus registers the effects of factor deficiencies just as the thrombin generation test does.

Example 4: Establishment of an Anticoagulatory Blood Coagulation System Status by Determining the Strength of Anticoagulation of a Factor Xa Inhibitor

[0119] The TF/lipid reagent acting as coagulation activator was prepared as follows: Innovin reagent (see example 1) was diluted 1:98.6 with Owren's Veronal Buffer. To 1493.3 μL of this dilution was added 6.7 μL of a phospholipid suspension (68% 1,2-dioleoyl-sn-glycero-3-phosphocholine, 32% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine, together 11.4 g/L).

[0120] This modified coagulation activator was used to perform the F1+2 generation test as described in example 2.

[0121] The anticoagulated samples were produced by same procedure as in the creation of the calibration curve for the Innovance anti-Xa rivaroxaban assay (Siemens Healthcare Diagnostics Products GmbH, Germany). A standard 0 (=normal plasma pool without rivaroxaban) and a standard 1 (normal plasma pool containing 410 ng/mL rivaroxaban) were mixed in various ratios.

[0122] The samples were measured using the Innovance anti-Xa rivaroxaban assay, a chromogenic anti-FXa test for determining the concentration of rivaroxaban, and with the inventive F1+2 generation test.

[0123] In the F1+2 generation test with an incubation time of 15 minutes after addition of CaCl.sub.2), a dependence of the signals (kcnt) on the rivaroxaban concentration very similar to that in the specific anti-Xa rivaroxaban test (mA signal) was observed (FIG. 6). The F1+2 generation test can thus likewise be used for quantitation of anticoagulants. Advantageously, the test can very readily achieve a higher sensitivity by shortening the incubation time or a broader measurement range by prolonging the incubation time. With an incubation time of 3 minutes (after addition of CaCl.sub.2)) the test responds very sensitively to an increase in the rivaroxaban concentration from 0 to 10 ng/mL. With an incubation time of 30 minutes (after addition of CaCl.sub.2)), the measurement range can be extended to over 400 ng/mL. Such a change to the incubation time can be made automatically by the analysis system in the form of a repeat measurement when the initial measurement result is below or above the measurement range.

Example 5: Establishment of an Anticoagulatory Blood Coagulation System Status by Determining the Strength of Anticoagulation of a Factor IIa Inhibitor

[0124] The TF/lipid reagent acting as coagulation activator was prepared as described in example 4, and the F1+2 generation test performed therewith as described in example 2.

[0125] The anticoagulated samples were produced by same procedure as in the creation of the calibration curve for the Innovance DTI assay (Siemens Healthcare Diagnostics Products GmbH, Germany). A standard 0 (=normal plasma pool without dabigatran) and a standard 1 (normal plasma pool containing 548 ng/mL dabigatran) were mixed in various ratios.

[0126] The samples were measured using the Innovance DTI assay, a chromogenic anti-FIIa test for determining the concentration of dabigatran.

[0127] In the F1+2 generation test with an incubation time of 5 minutes after addition of CaCl.sub.2), a dependence of the signals (kcnt) on the dabigatran concentration very similar to that in the specific anti-IIa dabigatran test (mA/min signal) was observed (FIG. 7). The F1+2 generation test can thus likewise be used for quantitation of anticoagulants. Advantageously, the test can very readily achieve a higher sensitivity by shortening the incubation time or a broader measurement range by prolonging the incubation time (after addition of CaCl.sub.2)). FIG. 8 shows the increase in signal height in the F1+2 generation test at a dabigatran concentration of 120 ng/mL resulting from an increase in incubation time. Such a change to the incubation time can be made automatically by the analysis system in the form of a repeat measurement when the initial measurement result is below or above the measurement range.

Example 6: Shortening the Incubation Time in the F1+2 Generation Test

[0128] The comparative determination of the amount of in-vitro-generated F1+2 and of the ETP was carried out as described in example 2, except that, in the F1+2 generation test, incubation after the addition of CaCl.sub.2) was for only 5 minutes instead of incubation for 20 minutes. The calibration and the production and measurement of the samples (samples with increasing coagulation factor deficiency in % of norm) was carried out as described in example 2.

[0129] The correlation with the classical ETP test of the results of the F1+2 generation test with incubation for 5 minutes remains linear (see FIG. 9). The slope of the lines (significantly greater than 1) points to a need for specific standardization. Since the decrease in the measured values shows the same linear relationship with the decrease in the coagulation factors, decreasing the incubation time is possible. This results in the overall time taken for the F1+2 generation test being shortened by 15 minutes, to 7.5 minutes, which represents a considerable advantage compared to the ETP test of the prior art.

Example 7: Establishment of a Procoagulatory Blood Coagulation System Status by Determining an Increase in Platelet Activation Through Measurement of the F1+2 Generation Potential

[0130] The detection of a hyper- or hypocoagulatory state arising from dysfunctional thrombin functionality or F1+2 generation functionality in blood cells is possible only when such blood cells are present in the sample. To detect dysfunction in platelet functionality, it is necessary to use either PRP or whole blood as the sample. To detect dysfunction in other blood cells, it is necessary to use whole blood as the sample.

[0131] A hypercoagulatory state in the sample (whole blood, PRP or plasma) was achieved by preactivating platelets with collagen. This was done by mixing 900 μL of sample with 100 μL of collagen solution (20 μg/mL), or with 100 μL of OVB buffer for the control, and incubating the mixture for 20 minutes at 37° C. in a water bath.

[0132] In order to be able to sensitively detect the influence of the blood cells, the Innovin reagent, which comprises tissue factor and lipid, was diluted 1:780 with OVB buffer.

[0133] The preactivated samples were mixed with the components of the Innovance ETP assay (Siemens Healthcare Diagnostics Products GmbH, Germany) as follows: [0134] 108 μL sample [0135] +40 μL ETP buffer [0136] +35 μL ETP reagent [0137] 150 seconds incubation [0138] +10 μL Innovin reagent (prediluted 1:780 with OVB) [0139] +14 μL ETP CaCl.sub.2) [0140] 30 minutes incubation at 37° C. [0141] 1:100 dilution of the reaction mixture with OVB buffer.

[0142] As in example 1, 5 μL of the diluted reaction mixture was used for determining the amount of F1+2 formed in the reaction mixture.

[0143] Three seemingly healthy blood donors were investigated. When using PRP or whole blood as sample, preactivation of the platelets could be demonstrated in whole blood and PRP by the substantial increase in F1+2 generation compared to non-preactivated platelets (addition of OVB buffer instead of collagen) (see FIG. 10). Measurement of the F1+2 generation potential in whole blood or PRP can thus be used to demonstrate hypercoagulability of cellular origin and thus a procoagulatory blood coagulation system status.

[0144] Blood donor 2 is unusual in having a particularly high F1+2 generation potential, which is already clear without collagen activation of the platelets and even in plasma as sample. This indicates the in-vivo presence in this donor of a procoagulatory blood coagulation system status.

Example 8: Establishment of an Anticoagulatory Blood Coagulation System Status by Determining the Anticoagulatory Effect of a Reduced Platelet Count

[0145] PRP and PPP samples from three apparently healthy blood donors were prepared and the platelet count determined.

[0146] The F1+2 generation potential was measured with PRP as sample and with and without preactivation through addition of collagen as described in example 7, the collagen solution here having a concentration of 80 μg/mL. The results of the samples from the three blood donors were averaged. Below a platelet count of <100 000/μL the F1+2 generation potential decreases when the platelets are not preactivated. When the platelets are preactivated, the F1+2 generation potential decreases below a platelet count of <50 000/μL (see FIG. 11).

[0147] Measurement of the F1+2 generation potential therefore indicates an anticoagulatory blood coagulation system status caused by thrombocytopenia. The results for the F1+2 generation reflects the situation in vivo, in which thrombocytopenia has less of an effect when the platelets present are particularly active.

Example 9: Establishment of an Procoagulatory Blood Coagulation System Status by Determining the Procoagulatory Effect of Lupus Anticoagulant

[0148] The most commonly used test for the determination of lupus anticoagulant (LA) is the DRVVT test (dilute Russell's viper venom test). In this test, coagulation is triggered using RVV as coagulation activator. First of all, a test is carried out in which the coagulation activator reagent has only a very low phospholipid content (LA1 test). If lupus anticoagulant is present, the coagulation time in the LA1 test is lengthened. This is followed by the performance of a second test (LA2) using a coagulation activator reagent that has a higher phospholipid concentration. The LA2 test also then shows an approximately normal coagulation time when lupus anticoagulant is present. A LA2/LA1 ratio for the two tests is close to 1 when a normal sample is measured and falls below 1 when lupus anticoagulant is present.

[0149] The lupus anticoagulant test was carried out in the standard manner but with the difference that, instead of the measurement of the coagulation time, the amount of F1+2 formed in the reaction mixture was determined (see example 1).

Performance of the LA1 Test:

[0150] 100 μL sample [0151] 240 seconds incubation at 37° C. [0152] +100 μL LA1 reagent [0153] 80 seconds incubation at 37° C. [0154] 1:50 dilution of the reaction mixture with OVB buffer.

Performance of the LA2 Test:

[0155] 100 μL sample [0156] 240 seconds incubation at 37° C. [0157] +100 μL LA2 reagent [0158] 80 seconds incubation at 37° C. [0159] 1:50 dilution of the reaction mixture with OVB buffer.

[0160] In each case 5 μL of the diluted reaction mixtures was used for determining the amount of F1+2 formed (see example 1).

[0161] A normal plasma sample without lupus anticoagulant (CPN), a sample with a high LA concentration (high LA control), and a sample with low LA concentration (low LA control) were tested, as were 1:1 dilutions of the high and low LA controls with normal plasma.

[0162] FIG. 12 compares the LA2/LA1 ratios in the coagulation test with the LA1/LA2 ratios in the F1+2 generation test. For the normal plasma all ratios are close to 1. The sample with the low lupus anticoagulant content (low LA control) has a LA1/LA2 ratio in the F1+2 generation test (0.21) that is considerably lower than the LA2/LA1 ratio in the coagulation test (0.80). The F1+2 generation test thus responds considerably more sensitively to a weakly pathological lupus anticoagulant content than does the coagulation test.

[0163] In the lupus anticoagulant test the sample is diluted with normal plasma when factor deficiency is to be excluded as the cause for pathological values. In the 1:1 mixture with normal plasma (CPN), the ratio falls only to 0.86 in the coagulation test in the case of the low LA control, whereas in the F1+2 generation test the ratio still falls to 0.50. Even in a 1:1 mixture with normal plasma, this means that a weakly presenting lupus anticoagulant can be detected considerably more readily by measurement with the F1+2 generation.

Example 10: Establishment of an Anticoagulatory Blood Coagulation System Status by Determining the Strength of Anticoagulation of a Platelet Inhibitor

[0164] In patients at increased risk of thrombosis, inhibition of platelets is indicated as prophylactic therapy. Such inhibition is accomplished in this example with prostaglandin E1, a substance that is known to inhibit platelet aggregation.

[0165] Whole blood from 3 normal and healthy blood donors was treated with 5% (v/v) of a prostaglandin E1 solution (end concentration in the sample 20 μmol/L) and incubated at room temperature for 20 minutes. This was followed by activation by collagen and further treatment and testing of the samples as described in example 7.

[0166] FIG. 13 shows the F1+2 generation potentials of samples with and without PGE1 pretreatment and with and without subsequent collagen activation. The anticoagulatory effect of the platelet inhibitor PGE1 is seen both without and with activation of platelets by collagen, in the form of decreased F1+2 generation. The test of embodiments of the present invention can be used for the monitoring of treatment with platelet inhibitors.

Example 11: Highly Sensitive Determination of Factor VIII Activity

[0167] Three samples having a known factor VIII activity were prepared by mixing a normal plasma pool having a known factor VIII activity (control plasma N) with Owren's Veronal Buffer. These samples are mixed with reagents as follows. All reagents are marketed by Siemens Healthcare Diagnostics Products GmbH, Germany. [0168] 2 μL sample [0169] +18 μL buffer [0170] 10 seconds incubation [0171] +15 μL factor-VIII-deficient plasma (<1% factor VIII) [0172] 120 seconds incubation [0173] +30 μL actin FS reagent (coagulation activator) [0174] 180 seconds incubation [0175] +40 μL CaCl.sub.2 solution [0176] 120 seconds incubation [0177] +15 μL H.sub.2O

[0178] As in example 1, 5 μL of the diluted reaction mixture was used for determining the amount of F1+2 formed in the reaction mixture.

[0179] After further incubation of the reaction run for a period of 6 minutes, the chemiluminescence signal in the arbitrary unit kilocounts (kcnt) was measured.

[0180] Results: [0181] Sample 1 0% factor VIII=1876 kcnt [0182] Sample 2 0.5% factor VIII=2469 kcnt [0183] Sample 3 1.0% factor VIII=2779 kcnt [0184] Sample 4 1.5% factor VIII=2901 kcnt

[0185] The F1+2 generation test permits a highly sensitive factor VIII activity test having large changes in signal in the range between 0 and 1.5% factor VIII activity.

[0186] The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” may be intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms “comprises,” “comprising,” “including,” and “having,” are inclusive and therefore specify the presence of stated features, elements, compositions, steps, integers, operations, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Although the open-ended term “comprising,” is to be understood as a non-restrictive term used to describe and claim various embodiments set forth herein, in certain aspects, the term may alternatively be understood to instead be a more limiting and restrictive term, such as “consisting of” or “consisting essentially of.” Thus, for any given embodiment reciting compositions, materials, components, elements, features, integers, operations, and/or process steps, the present disclosure also specifically includes embodiments consisting of, or consisting essentially of, such recited compositions, materials, components, elements, features, integers, operations, and/or process steps. In the case of “consisting of,” the alternative embodiment excludes any additional compositions, materials, components, elements, features, integers, operations, and/or process steps, while in the case of “consisting essentially of,” any additional compositions, materials, components, elements, features, integers, operations, and/or process steps that materially affect the basic and novel characteristics are excluded from such an embodiment, but any compositions, materials, components, elements, features, integers, operations, and/or process steps that do not materially affect the basic and novel characteristics can be included in the embodiment.

[0187] In addition, disclosure of ranges includes disclosure of all values and further divided ranges within the entire range, including endpoints and sub-ranges given for the ranges. Thus ranges are, unless specified otherwise, inclusive of endpoints and include disclosure of all distinct values and further divided ranges within the entire range. Disclosure of values and ranges of values for specific parameters (such as temperatures, molecular weights, weight percentages, etc.) are not exclusive of other values and ranges of values useful herein. It is envisioned that two or more specific exemplified values for a given parameter may define endpoints for a range of values that may be claimed for the parameter. For example, if Parameter X is exemplified herein to have value A and also exemplified to have value Z, it is envisioned that Parameter X may have a range of values from about A to about Z. Similarly, it is envisioned that disclosure of two or more ranges of values for a parameter (whether such ranges are nested, overlapping or distinct) subsume all possible combination of ranges for the value that might be claimed using endpoints of the disclosed ranges. For example, if Parameter X is exemplified herein to have values in the range of 1-10, or 2-9, or 3-8, it is also envisioned that Parameter X may have other ranges of values including 1-9, 1-8, 1-3, 1-2, 2-10, 2-8, 2-3, 3-10, and 3-9.

[0188] Although the present invention has been shown and described with respect to certain example embodiments, equivalents and modifications will occur to others skilled in the art upon the reading and understanding of the specification. The present invention includes all such equivalents and modifications and is limited only by the scope of the appended claims.