Method For Determining Lupus Anticoagulant In A Single Coagulation Reaction

Abstract

The invention is in the field of coagulation diagnostics and relates to a method for detecting lupus anticoagulant.

Claims

1. A method for detecting lupus anticoagulant in a plasma sample of a patient, the method comprising the steps of: providing a reaction mixture by addition of a lupus anticoagulant-sensitive activated partial thromboplastin time (APTT) reagent to the plasma sample and starting the coagulation reaction, measuring a measurement variable S of the reaction mixture over time, resulting in a function S(t) of time-dependent measurement values, and determining the coagulation time, characterized in that a) the maximum reaction velocity v.sub.max and/or the maximum reaction acceleration a.sub.max of the function S(t) are determined, and b) the absolute value of the difference between a first measurement value SB at the start of measurement and a second measurement value SE at the end of measurement (|DeltaS|) is ascertained, and then c) the relative maximum reaction velocity v.sub.max rel and/or the relative maximum reaction acceleration a.sub.max rel are determined using the following formulae: $v_{\max \mathrm{rel}}=v_{\max }/\left|\mathrm{Delta}S\right|\mathrm{and}$ $a_{\max \mathrm{rel}}=a_{\max }/\left|\mathrm{Delta}S\right|,$ respectively, lupus anticoagulant is detected if the coagulation time is prolonged compared to a predetermined reference value and the relative maximum reaction velocity v.sub.max rel and/or the relative maximum reaction acceleration a.sub.max rel are within a predetermined lupus anticoagulant-specific range of values.

2. The method according to claim 1, wherein the lupus anticoagulant-sensitive APTT reagent contains phospholipids, a contact activator and optionally calcium ions.

3. The method according to claim 1, wherein the maximum reaction velocity v.sub.max corresponds to the maximum or the minimum of the first derivative of the function S(t) of the time-dependent measurement values.

4. The method according to claim 1, wherein the maximum reaction acceleration a.sub.max corresponds to the maximum or the minimum of the second derivative of the function S(t) of the time-dependent measurement values.

5. An automatic analyser comprising (i) one or more pipetting devices for transfer of a sample volume and at least one reagent volume into a reaction vessel for preparation of a reaction mixture, (ii) a measurement device for measurement of a measurement variable S of the reaction mixture in the reaction vessel over time (t), (iii) a data memory for storage of a function S(t) of time-dependent measurement values which were measured for a sample or a reaction mixture, and (iv) an evaluation device configured such that it uses the function S(t) of time-dependent measurement values from the data memory for calculation of a coagulation time, characterized in that the evaluation device is additionally configured such that it a) determines the maximum reaction velocity v.sub.max and/or the maximum reaction acceleration a.sub.max of the function S(t), and b) ascertains the absolute value of the difference between a first measurement value SB at the start of measurement and a second measurement value SE at the end of measurement (|DeltaS|), and then c) determines the relative maximum reaction velocity v.sub.max rel and/or the relative maximum reaction acceleration a.sub.max rel using the following formulae: $v_{\max \mathrm{rel}}=v_{\max }/\left|\mathrm{Delta}S\right|\mathrm{and}$ $a_{\max \mathrm{rel}}=a_{\max }/\left|\mathrm{Delta}S\right|,$ respectively, d) compares the determined coagulation time with a predetermined reference value, and e) compares the relative maximum reaction velocity v.sub.max rel and/or the relative maximum reaction acceleration a.sub.max rel with a respectively predetermined lupus anticoagulant-specific range of values, and f) outputs the presence of lupus anticoagulant in the sample as the result if the coagulation time is prolonged compared to the predetermined reference value and the relative maximum reaction velocity v.sub.max rel and/or the relative maximum reaction acceleration a.sub.max rel are within the respective predetermined lupus anticoagulant-specific range of values.

6. The automatic analyser according to claim 5, in which the measurement device is suitable for measurement of an optical measurement variable S of a reaction mixture.

7. The automatic analyser according to claim 6, in which the measurement device for measurement of an optical measurement variable S of a reaction mixture is a photometer.

Description

DESCRIPTION OF FIGURE

[0041] FIG. 1 shows a typical APTT coagulation curve on a Sysmex CS-2100 analyser. Curve 1 shows the optical transmissivity/transmission (S) of the reaction mixture in artificial units [AU] over time, over a period of 180 s. The difference Delta S (ΔS) between the transmissivities at the end of measurement (measurement value SE at time point t=180 s) and at the start of measurement (measurement value SB at time point t=0 s) can be read in the software. The APTT coagulation time is determined by determining the time point in which the measurement value corresponds to 50% of the difference Delta S (ΔS). Curve 2 shows the numerical 1st derivative with respect to time (dS/dt), which is a measure of the velocity v of the reaction. It has a minimum at the highest reaction velocity v.sub.max. Curve 3 shows the numerical 2nd derivative with respect to time (d.sup.2S/dt.sup.2), which is a measure of the acceleration a of the reaction. It has a minimum at the highest reaction acceleration a.sub.max—The numerical values for the maximum reaction velocity v.sub.max and the maximum reaction acceleration a.sub.max can be read via the software. The numerical values for the relative maximum velocity V.sub.max rel and the relative maximum acceleration a.sub.max rel are yielded by division of the absolute values for maximum velocity and maximum acceleration by the absolute value of Delta S.

[0042] The invention will be elucidated below on the basis of an exemplary embodiment.

EXAMPLE: Identification of Lupus Anticoagulant-Positive Samples in a Single APTT Coagulation Mix in Each Case in Accordance with the Invention

[0043] The following types of low-platelet-count plasma samples were tested:

TABLE-US-00001 Sample type Description Normal Pool of >100 donations from healthy individuals, stabilized; Heparin Pool of >100 donations from healthy individuals, stabilized and admixed with unfractionated or low-molecular- weight heparin (UF or LMW heparin); Factor VIII Mixture of factor VIII-deficient plasma deficiency and normal pool; Lupus Plasmas from individual donors, lupus anticoagulant anticoagulant-positive in a test system (LA) based on the dilute Russell's viper venom (DRVVT) test.

[0044] For the measurement of APTT, the test setting approved on the Sysmex CS-2100 analyser (Sysmex Corp.) is used. 50 μL of sample are mixed with 50 μL of lupus anticoagulant-sensitive APTT reagent (Actin FSL reagent, Siemens Healthcare Diagnostics Products GmbH) containing phospholipids and ellagic acid as activator. After 180 s of incubation at +37° C., 50 μL of 25 mM CaCl.sub.2 are added to start the reaction, and the acquisition of measurement values is started.

[0045] The APTT coagulation time is determined by ascertaining the time point at which the measured optical transmissivity of the reaction mixture is 50% of the difference Delta S between the transmissivities at the end of measurement (measurement value SE at time point t=180 s) and at the start of measurement (measurement value SB at time point t=0 s). Coagulation time results of more than 30 s are considered prolonged.

[0046] The relative maximum reaction velocity v.sub.max rel is determined by first ascertaining the maximum reaction velocity v.sub.max by determination of the minimum of the 1st derivative of the reaction kinetics with respect to time (dS/dt) and lastly dividing v.sub.max by the absolute value of Delta S.

[0047] The relative maximum reaction acceleration a.sub.max rel is determined by first ascertaining the maximum reaction acceleration a.sub.max by determination of the minimum of the 2nd derivative of the reaction kinetics with respect to time (d.sup.2S/dt.sup.2) and lastly dividing a.sub.max by the absolute value of Delta S.

[0048] The results for each sample are shown in Table 1.

TABLE-US-00002 TABLE 1 APTT v.sub.max rel a.sub.max rel Sample [s] [10.sup.−3 s.sup.−1] [10.sup.−5 s.sup.−2] Normal 1 24.8 7.0 11.3 Normal 2 27.3 6.9 11.1 LMW heparin, 1.0 IU/mL 50.4 3.9 5.2 UF heparin, 0.6 IU/mL 75.2 2.7 2.7 Factor VIII deficiency 1, 5% 46.7 2.7 2.8 Factor VIII deficiency 2, 5% 43.2 3.7 4.6 LA 1 51.6 1.3 1.5 LA 2 40.9 1.7 2.3 LA 3 37.4 1.2 1.6 LA 4 36.7 1.2 1.7 LA 5 31.7 1.1 1.6 LA 6 35.8 1.5 2.2 LA 7 36.7 1.2 1.5 LA 8 80.2 2.3 2.3 LA 9 69.1 1.9 2.0 LA 10 51.9 1.1 1.3

[0049] As expected, prolonged coagulation times (>30 s) are exhibited by heparin samples, factor VIII-deficient samples (having a deficiency of a factor of the intrinsic system) and lupus anticoagulant-positive samples, whereas normal samples do not exhibit prolonged coagulation times. As screening test for lupus anticoagulant, the APTT in this experiment has a sensitivity of 100%.

[0050] For lupus anticoagulant-positive samples, it becomes apparent, furthermore, that the above-described parameter v.sub.max rel is in a range of values from 1.1 to 2.3 10.sup.−3s.sup.−1, which range is specific for lupus anticoagulant-positive samples. Furthermore, it becomes apparent for lupus anticoagulant-positive samples that the parameter a.sub.max rel, likewise described above, is in a range of values from 1.3 to 2.3 10.sup.−5s.sup.−2, which range is likewise specific for lupus anticoagulant-positive samples. Other samples (normal samples, heparin samples, factor VIII-deficient samples) each have greater numerical values for the parameters v.sub.max rel and a.sub.max rel, which numerical values are outside the lupus anticoagulant-specific ranges of values for v.sub.max rel and a.sub.max rel.

[0051] Therefore, a two-step method which originates from only a single APTT measurement and comprises the determination of coagulation time and the determination of the parameters v.sub.max rel and/or a.sub.max rel is suitable as proof for lupus anticoagulant.

[0052] In the present experiment, the method for detecting lupus anticoagulant has a sensitivity of 100% and a specificity of 100%.