LUPUS ANTICOAGULANT (LA) MIXING METHOD AND KIT WITH REDUCED FACTOR DEFICIENCY EFFECT AND REDUCED INHIBITOR INTERFERENCE FOR IDENTIFYING LA ASSOCIATED WITH ANTIPHOSPHOLIPID SYNDROME

Abstract

The invention described herein relates to a method and kit for the diagnosis of antiphospholipid syndrome in patients, wherein a coagulation inhibitor and normal pooled plasma mixture reduce the factor deficiency effect of patient plasma, and coagulation inhibitor interference in patient plasma, and increases specificity and the sensitivity for detecting lupus anticoagulants in the patient's blood in a phospholipid-dependent clotting assay.

Claims

1. A method for detecting lupus anticoagulants associated with antiphospholipid syndrome in a patient, comprising: a) detecting the clotting time of a plasma sample from said patient in a phospholipid-dependent clotting assay measured in the presence of at least one coagulation inhibitor and a low concentration of phospholipid; b) detecting the clotting time of the plasma sample in the phospholipid clotting assay measured in the presence of the at least one coagulation inhibitor and a concentration of phospholipid higher than said low concentration of phospholipid in step a); c) determining a ratio of the clotting time of the patient plasma in step a) to the clotting time of the patient plasma in step b), and comparing the patient ratio to a cutoff ratio established from normal, healthy subjects.

2. The method of claim 1 wherein each of the phospholipid-dependent clotting assays of step a) and the phospholipid-dependent clotting assay of step b) further comprise normal pooled plasma.

3. The method of claim 1 wherein the phospholipid-dependent assay comprises activated partial thromboplastin time (APTT).

4. The method of claim 1 wherein the phospholipid-dependent assay is selected from the group consisting of dilute Russell's viper venom time (dRVVT), APTT-based platelet neutralization procedure (PNP), APTT-based hexagonal phase phospholipid neutralization test, APTT-based kaolin clotting time (KCT), APTT-based silica clotting time (SCT) and dilute prothrombin time (dPT).

5. The method of claim 1 wherein at least one coagulation inhibitor comprises at least one thrombin inhibitor.

6. The method of claim 5 further comprising selecting at least one thrombin inhibitor from the group consisting of d-phenylalanyl-1-prolyl-1-arginine-chloromethyl ketone (PPACK), I-2581, hirudin, hirudin derivatives, lepirudin, desirudin, hirudin analogues, bivalirudin, argatroban, melagatran, dabigatran, and combinations thereof.

7. The method of claim 1 wherein the coagulation inhibitor comprises at least one factor Xa inhibitor selected from the group consisting of rivaroxaban, apixaban, edoxaban, betrixaban, antistasin, Tick Anticoagulant Protein (TAP), and combinations thereof.

8. The method of claim 2 further comprising diluting said patient plasma sample with normal pooled plasma.

9. The method of claim 2 further comprising diluting said patient plasma sample 1:1 with normal pooled plasma.

10. The method of claim 2 further comprising diluting said patient plasma sample 1:2, 1:3 or 1:4 with said normal pooled plasma.

11. The method of claim 1 wherein said low concentration of phospholipid comprises a range of about 0.002 (g/l) to about 0.2 (g/l).

12. The method of claim 1 wherein said high concentration of phospholipid comprises a range of about 0.2 (g/l) to about 5.0 (g/l).

13. A kit for detecting lupus anticoagulants associated with antiphospholipid syndrome in a patient, comprising: a) a phospholipid-dependent clotting reagent comprising a low concentration of phospholipid; b) at least one coagulation inhibitor; and, c) said phospholipid-dependent clotting reagent comprising a concentration of phospholipid that is higher than the concentration of phospholipids in a).

14. The kit of claim 13 further comprising normal pooled plasma.

15. The kit of claim 13 wherein said phospholipid-dependent clotting reagent comprises APTT.

16. The kit of claim 13 wherein said phospholipid-dependent clotting reagent is selected from the group consisting of dRVVT, APTT-based platelet neutralization procedure (PNP), APTT-based hexagonal phase phospholipid neutralization test, APTT-based kaolin clotting time (KCT), APTT-based silica clotting time (SCT) and dilute prothrombin time (dPT).

17. The kit of claim 13 wherein said at least one coagulation inhibitor comprises at least one thrombin inhibitor.

18. The kit of claim 17 wherein said at least one thrombin inhibitor is selected from the group consisting of d-phenylalanyl-1-prolyl-1-arginine-chloromethyl ketone (PPACK), I-2581, hirudin, hirudin derivatives, lepirudin, desirudin, hirudin analogues, bivalirudin, argatroban, melagatran, dabigatran, and combinations thereof.

19. The kit of claim 13 wherein said at least one coagulation inhibitor comprises at least one factor Xa inhibitor, selected from the group consisting of rivaroxaban, apixaban, edoxaban, betrixaban, antistasin, Tick Anticoagulant Protein (TAP) and combinations thereof

20. The kit of claim 13 wherein said coagulation inhibitor and said phospholipid-dependent clotting reagent comprising said low concentration of phospholipid are packaged in the same container.

21. The kit of claim 13 wherein said coagulation inhibitor and said phospholipid-dependent clotting reagent comprising said high concentration of phospholipid are packaged in the same container.

22. The kit of claim 14 wherein said normal pooled plasma is packaged in the same container as said coagulation inhibitor.

23. The method of claim 1 wherein the coagulation inhibitor is a combination of a thrombin inhibitor and a non-thrombin coagulation inhibitor.

24. The method of claim 23 wherein the non-thrombin inhibitor comprises a factor Xa inhibitor.

25. The method of claim 2 wherein the coagulation inhibitor and normal pooled plasma mixture reduces interference caused by the presence in the plasma of a factor deficiency or a coagulation inhibitor and increases specificity and the sensitivity of the method for detecting lupus anticoagulants.

26. A kit for detecting lupus anticoagulants associated with antiphospholipid syndrome in a patient, comprising: at least one coagulation inhibitor; and normal pooled plasma.

27. The method of claim 1 wherein said cutoff ratio is a ratio of NR between about 1.00 and about 1.20.

28. The kit of claim 13 wherein said low concentration of phospholipid comprises a range of about 0.002 (g/l) to about 0.2 (g/l).

29. The kit of claim 13 wherein said higher concentration of phospholipid comprises a range of about 0.2 (g/l) to about 5.0 (g/l).

Description

BRIEF DESCRIPTION OF THE FIGURES

[0040] FIGS. 1A-B illustrate the effect of d-phenylalanyl-1-prolyl-1-arginine-chloromethyl ketone (PPACK) concentrations on the clotting time for A PTT-based silica clotting (SCT Screen) and APTT-based silica clotting time confirm (SCT Confirm) according to one embodiment of the invention. PPACK at increasing concentrations showed different effects on the SCT Screen Test of normal plasma samples and LA positive plasma samples but not on the SCT Confirm Test.

[0041] FIG. 2 illustrates in graph form the effect of PPACK concentrations on the Screen-to-Confirm Ratio of the SCT Assay according to one embodiment of the invention. Increasing PPACK concentrations increased the Screen-to-Confirm Ratio of LA positive sample from 1.09 to 1.82. The Screen-to-Confirm Ratio of normal pooled plasma (NPP) remained the same.

[0042] FIG. 3 illustrates in graph form the effect of PPACK concentration on the Normalized Ratio (NR) of S(CT Assay according to one embodiment of the invention. The LA positive sample at 3 concentrations (undiluted, 1:1 or 1:3 diluted with NPP), had a NR of 2.34, 1.82, and 1.46, respectively at 1000 ng/ml of PPACK in the automated mixing study (1:1 dilution with NPP/PPACK material). Even the most dilute sample (an effective eight fold dilution of the original sample with NPP) still remains as a positive LA result.

[0043] FIGS. 4A-4B illustrate in graph form the effect of PPACK concentration on the clotting time for dRVVT Screen test and dRVVT Confirm test according to one embodiment of the invention. FIG. 4A illustrates that PPACK at increasing concentrations showed different effects on the dRVVT Screen test of normal plasma samples and LA positive plasma samples. FIG. 4B illustrates that PPACK at increasing concentrations did not show different effects on the dRVVT Confirm test.

[0044] FIG. 5 illustrates in graph form the effect of PPACK concentration on the Screen-to-Confirm Ratio of dRVVT test. Increasing PPACK concentrations increased the Screen-to-Confirm Ratio of the LA positive sample from 1.42 to 1.58, while it decreased the Screen-to-Confirm ratio of NPP from 1.02 to 0.91.

[0045] FIG. 6 illustrates in graph form the effect of PPACK concentrations on the NR of dRVVT test. The LA positive sample at 3 concentrations (undiluted, 1:1 or 1:3 diluted with NPP), had an NR of 1.76, 1.42 and 1.19, respectively at 1000 ng/ml PPACK in the automated mixing study (1:1 dilution with NPP/PPACK material). Even the most dilute sample (an effective eight fold dilution of the original sample with NPP) still remains as a positive LA result.

[0046] FIGS. 7A-B illustrate in graph form the effect of I-2581 and the combined use of I-2581 and PPACK on the Screen-to-Confirm Ratio and the NR of the dRVVT Assay according to one embodiment of the invention. FIG. 7A illustrates that I-2581 had similar impact on the Screen-to-Confirm Ratio of LA positive and NPP samples in the absence of PPACK with both slightly decreased at the presence of I-2581. At the presence of 350 nM PPACK, the effect from I-2581 on the Screen-to-Confirm Ratio remained similar for LA positive and NPP samples.

[0047] FIG. 7B illustrates that I-2581 contributed to a slight decrease in the normalized ratio with or without the combined use of PPACK.

[0048] FIGS. 8A-B illustrate in graph form the effect of hirudin, argatroban and their combined use on the Screen-to-Confirm Ratio of the dRVVT Assay according to one embodiment of the invention. FIG. 8A illustrates that hirudin increased the Screen-to-Confirm Ratio for both NPP (from 1.02 in the absence of hirudin to 1.18 at 1000 ng/ml hirudin) and LA positive sample (from 1.41 in the absence of hirudin to 2.14 at 1000 ng/ml hirudin), although the effect on NPP sample was rather small. FIG. 8B illustrates that when argatroban was used as the inhibitor, the Screen-to-Confirm Ratio responded in a different pattern: the ratio for LA positive sample (squares) didn't change, while the ratio for NPP (crosses) decreased noticeably (from 1.06 in the absence of argatroban to 0.9 at 500 ng/ml argatroban). Hirudin and argatroban displayed a synergistic effect when used together (triangles for NPP and diamonds for LA positive sample). A synergistic effect may occur with the combination of other coagulation inhibitors, e.g., PPACK and rivaroxaban.

[0049] FIGS. 9A-B illustrate in graph form the effect of hirudin, argatroban and their combined use on the normalized ratio of the dRVVT Assay according to one embodiment of the invention. FIG. 9A illustrates the normalized ratio increased from 1.39 (without thrombin inhibitor use) to 1.81 (at 1000 ng/ml hirudin) for the LA positive sample. FIG. 9B illustrates that argatroban increased the Normalized Ratio from 1.34 to 1.57 (at 500 ng/ml argatroban, squares) by decreasing the NPP Screen-to-Confirm Ratio. When hirudin and argatroban were used together, the normalized ratio decreased from 1.72 (200 ng/ml hirudin alone) to 1.66 (200 ng/ml hirudin and 500 ng/ml argatroban) for the LA positive sample (diamonds).

DESCRIPTION OF THE INVENTION

[0050] Anticoagulants against thrombin and factor Xa prolong clotting times in LA assays by effectively reducing the available amounts of those factors in the reaction mixture. However, the effect from the anticoagulants on LA negative and LA positive plasma specimens differs. Coagulation inhibitors, e.g., thrombin and factor Xa inhibitors, in an LA assay increases sensitivity to LA antibodies, which allows reliable mixing studies to be performed to address the interference caused by factor deficiency or endogenous coagulation factors. The difference provides methods for improving the interpretation of LA assays both when the test plasma sample is diluted with normal plasma to normalize clotting factors and when it is not. Therefore, LA sensitivity, for example, detecting weak LA positive plasma, can be significantly increased using reagents with anticoagulant added without sacrificing the LA assay specificity. Further, the invention described herein boosts the LA signal in so-called “mixing assays”, discussed above, for detecting LA.

[0051] The current invention employs a methodology whereby selected anticoagulants are added to LA assays to achieve improved assay sensitivity and specificity and to render a more reliable automated mixing study (adding normal plasma substantially free of platelets (NPP) to the patient plasma sample and repeating the clotting assay) to address factor deficiency and factor inhibitor problems in LA detection for the diagnosis of APS. Specifically, according to the invention described herein, the synergistic effects from different inhibitors including but not limited to thrombin inhibitors PPACK, hirudin, hirudin derivatives, e.g., lepirudin and desaridin, hirudin analogues, e.g., bivaluridin, argatroban, melagatran, dabigatran, I-2581, and their combinations, when added to standard phospholipid-dependent assays provide unique tools in optimizing the sensitivity and specificity in LA detection and diagnosis of APS. The amount of coagulation inhibitor added must be optimized to increase the clotting time ratio of LA positive samples while continuing the correction of the LA negative samples that have factor deficiencies or factor inhibitors that are corrected by the mixing study plasma added to the sample.

[0052] According to one aspect, the invention is a kit for aiding in the detection of lupus anticoagulants for the diagnosis of APS in a patient. In one embodiment of the invention, the kit includes at least one coagulation inhibitor, e.g., such as but not limited to a thrombin inhibitor, for example, but not limited to the thrombin inhibitors detailed above, and normal plasma, pooled or otherwise. In another embodiment, the kit above further includes, or is intended to be used with an LA assay such as an APTT reagent, for example, APTT-based silica clotting time (SCT), APTT-based platelet neutralization procedure (PNP), APTT-based hexagonal phase phospholipid neutralization test, APTT-based kaolin clotting time (KCT), having a low phospholipid concentration, in the range of 0.002 (g/l) to 0.2 (g/l), preferably 0.01 (g/l), and the LA assay having a high phospholipid concentration, in the range of 0.2 (g/l) to 5.0 (g/l), preferably 1.0 (g/l).

[0053] In another aspect, the invention is a method for detecting lupus anticoagulants to aid in the diagnosis of APS in a patient. In one embodiment of the method of the invention, a coagulation screen clotting assay is conducted by mixing a patient's plasma with one or more coagulation inhibitors, a thrombin inhibitor, for example, one or more of the thrombin inhibitors or factor Xa inhibitors above, and a phospholipid-dependent APTT reagent with low phospholipid concentration. The screen clotting time is measured. A coagulation confirm clotting assay is also done on the patient's plasma by mixing the patient's sample with the thrombin inhibitor used in the screen assay, and the phospholipid in the presence of a high concentration phospholipid APTT reagent, and the confirm clotting time is measured.

[0054] In an alternative embodiment, for example, high and low phospholipid APTT is replaced with high and low phospholipid as disclosed above in a dRVVT assay or dilute Prothrombin Time assay in the screen and confirm clotting assays of the patient's plasma.

[0055] In another embodiment of the method of the invention, for example, a factor Xa screen clotting assay is conducted by mixing a patient's plasma with a factor Xa inhibitor, for example, but not limited to, rivaroxaban, apixaban, edoxaban, betrixaban, antistasin, Tick Anticoagulant Protein (TAP) and combinations thereof and an APTT reagent with low phospholipid concentration as described above. The clotting time is measured. A confirm clotting assay is also done on the patient's plasma by mixing the patient's sample with the factor Xa inhibitor used in the factor Xa screen assay of the patient's plasma sample, and the high phospholipid concentration APTT reagent as described above APTT reagent. The confirmed clotting time is measured.

[0056] In an alternative embodiment, high and low phospholipid APTT is replaced with high and low phospholipid dRVVT in the screen and confirm clotting assays of the patient's plasma.

[0057] For each of the assays above, irrespective of the clotting inhibitor used in the assay, the measured screen assay clotting time of a patient sample is compared to the measured screen assay clotting time of normal platelet poor plasma and expressed as a ratio (Rs). Additionally, the measured confirm assay clotting time of a patient sample is compared to the measured confirm assay clotting time of normal platelet poor plasma and expressed as a ratio (Rc). A normalized ratio (NR) is expressed as Rs/Rc. The NR is compared to a cutoff ratio established from normal, healthy subjects.

[0058] In a preferred embodiment of the invention, the cutoff ratio is derived from preferably at least 40 normal, healthy subjects. The cutoff value is established as suggested in current LA guidelines as either the value above the 99.sup.th percentile of the distribution of normal samples or as the mean +2SD interval of the parametric distribution of normal samples. A test plasma ratio being greater than the cutoff ratio is indicative of the presence of lupus anticoagulants in the test plasma and repeat testing is suggested after 12 weeks to confirm persistent presence of circulating antiphospholipid antibodies.

[0059] In a preferred embodiment of the invention, the exemplary cutoff ratio is expressed as a ratio of NR between about 1.00 and about 1.20, preferably between about 1.00 and about 1.15, more preferably between about 1.00 and about 1.10.

[0060] In a preferred embodiment of the invention, the clotting time of normal platelet poor plasma is established from at least 40 normal, healthy subjects. The average clotting time of the normal, healthy subjects is used to normalize the test plasma clotting time.

[0061] In an alternative embodiment, the clotting time of normal platelet poor plasma is established from a pool of normal, healthy subjects. The clotting time of the pooled normal, healthy subjects is used to normalize the test plasma clotting time.

Exemplary Studies

[0062] D-phenylalanyl-1-prolyl-1-arginine-chloromethyl ketone (PPACK) is a synthetic peptide that irreversibly binds to the catalytic site of thrombin. Referring to FIGS. 1-3, the use of PPACK in an SCT assay is shown where a plasma test sample was mixed with a LA negative plasma sample pool in a 1:1 ratio during testing (i.e., in a mixing study setting). By a mixing study, clotting factors that are present in normal plasma are added to the test plasma suspected of having a coagulation disorder. If the coagulation abnormality (prolongation of clotting time) is corrected, the test plasma is missing a clotting factor and does not have LA.

[0063] The introduction of PPACK in an LA assay causes the prolongation of the clotting time for both SCT Screen and SCT Confirm, however, the effect of PPACK on SCT Screen (low phospholipid concentration) and SCT Confirm (higher phospholipid concentration) assays differs significantly for LA positive and LA negative (platelet poor NPP) samples. Referring to FIGS. 1A-B, while the effect of PPACK on SCT Screen and SCT Confirm is similar for the LA negative sample, PPACK has much greater impact on SCT Screen than on SCT Confirm for the LA positive samples. Thus, referring to FIG. 2, for the LA negative sample, the screen-to-confirm ratio decreased slightly at high PPACK concentrations (R was 0.81, 0.83 and 0.78 at 0, 100 and 1000 nM PPACK, respectively), and for the LA positive sample, the screen-to-confirm ratio increased from 1.09 in the absence of PPACK to 1.82 at 1000 nM PPACK, resulting in normalized ratios in the range of 1.36 to 2.34 for the LA positive sample when normalized to the LA negative sample at the same PPACK concentration (see FIG. 3).

[0064] The effect of PPACK on LA detection is further analyzed on the LA positive sample diluted with LA negative sample in different ratios (1:1 and 1:3 of LA positive: LA Negative). When the LA positive sample was diluted 4-fold with LA negative sample, the LA was not detectable in the absence of PPACK (NR: 1.08) but became clearly LA positive in the presence of 1000 nM PPACK (NR: 1.46) thereby underscoring the increased sensitivity of the method disclosed herein.

[0065] Referring now to FIGS. 4-6, the PPACK model demonstrates the same use of thrombin inhibitors on the dRVVT test, where test samples were mixed and diluted with LA negative sample in a 1:1 ratio during testing (i.e., in a mixing study setting).

[0066] The use of PPACK in the SCT assay and dRVVT assay demonstrates that much enhanced assay sensitivity may be achieved even when the sample is diluted in a mixing study, thus making the mixing study more useful in detecting LA when the complicating effects from factor deficiency and factor inhibitors are minimized by the addition of platelet poor normal plasma. The observed decrease in normalized ratio for LA negative samples also indicates that a slight improvement in assay specificity is likely.

[0067] When patient plasma samples (n=18) with various factor deficiencies (LA negative, factor V deficient, factor VIII deficient, factor VII deficient, factor VIII/von Willebrand factor deficient, factor IX deficient, factor X deficient, factor XI deficient, factor XII deficient, and LA positive plasma samples) were tested with HemosIL® dRVVT assay (Table 1) (no PPACK), two plasma samples with factor V deficiency and one sample with factor X deficiency showed high levels of screen ratio (Rs). Among these three abnormal plasma samples exhibiting a clotting factor deficiency, two samples failed the dRVVT Confirm test. Accordingly, for these two failed plasma samples, the normalized ratio (NR) could not be calculated.

TABLE-US-00001 TABLE 1 Patient Samples Assayed by HemosIL ® dRVVT Screen and dRVVT Confirm dRVVT Sample ID Description Screen, s Confirm, s S/C (R) Screen (Rs) Confirm (Rc) S/C (NR) 0114 LA NEG LA Negative Pool 36.4 35.3 1.03 1.00 1.00 1.00 0114 S01 FVDef (con) 155.9 Failed N/A 4.28 N/A N/A 0114 S02 FVDef (con) 57.5 59.9 0.96 1.58 1.70 0.93 0114 S03 FVIIIDef (con) 34.2 34.0 1.01 0.94 0.96 0.98 0114 S04 FVIIIDef (con) 34.7 33.2 1.05 0.95 0.94 1.01 0114 S05 FVIII (con)/vWF Def 34.0 33.7 1.01 0.93 0.95 0.98 0114 S06 FVIII (con)/vWF Def 33.8 33.4 1.01 0.93 0.95 0.98 0114 S07 FIXDef (con) 36.1 35.4 1.02 0.99 1.00 0.99 0114 S08 FIXDef (con) 34.4 33.0 1.04 0.95 0.93 1.01 0114 S09 FXDef (con) 216.0 Failed N/A 5.93 N/A N/A 0114 S10 FXDef (con) 52.9 49.6 1.07 1.45 1.41 1.03 0114 S11 FXIDef (con) 37.2 35.2 1.06 1.02 1.00 1.02 0114 S12 FXIDef (con) 32.7 33.5 0.98 0.90 0.95 0.95 0114 S13 FXIIDef (con) 31.5 31.1 1.01 0.87 0.88 0.98 0114 S14 FXIIDef (acq) 38.4 40.0 0.96 1.05 1.13 0.93 0114 S15 LA Postive 86.1 35.8 2.41 2.37 1.01 2.33 0114 S16 LA Postive 105.2 37.8 2.78 2.89 1.07 2.70 0114 S17 LA Postive 78.6 36.4 2.16 2.16 1.03 2.09

[0068] Table 2 results disclose that when the above plasma samples (LA negative, factor V deficient, factor VIII deficient, factor VII deficient, factor VIII/von Willebrand factor deficient, factor IX deficient, factor X deficient, factor XI deficient, factor XII deficient, and LA positive plasma samples) were tested with dRVVT in the presence of 35) nM PPACK in a mixing study setting (1:1 test plasma sample to NPP ratio), only one factor V deficiency sample showed slightly higher screen ratio (1.28 with PPACK vs 4.28 with HemosIL® dRVVT Screen). The study reported the normalized ratio correctly for all plasma samples and addressed issues with factor deficiency (Table 2).

TABLE-US-00002 TABLE 2 Patient Samples Assayed by HemosIL ® dRVVT Screen and dRVVT Confirm in the Presence of PPACK with a 1:1 Mixing Study Setting 0 nM PPACK 350 nM PPACK Screen, Confirm, S/C Screen Confirm S/C Screen, Confirm, S/C Screen Confirm S/C Sample ID Description s s (R) (Rs) (Rc) (NR) s s (R) (Rs) (Rc) (NR) 0114 LA NEG LA Negative Pool 35.8 34.6 1.03 1.0 1.0 1.0 49.6 50.4 0.98 1.00 1.00 1.00 0114 S01 FVDef (con) 43.4 39.1 1.11 1.21 1.13 1.07 63.3 62.8 1.01 1.28 1.25 1.02 0114 S02 FVDef (con) 33.7 36.1 0.93 0.94 1.04 0.90 46.9 54.2 0.87 0.95 1.08 0.88 0114 S03 FVIIIDef (con) 34.3 33.2 1.03 0.96 0.96 1.00 49.7 47.9 1.04 1.00 0.95 1.05 0114 S04 FVIIIDef (con) 34.4 32.5 1.06 0.96 0.94 1.02 49.3 48.5 1.02 0.99 0.96 1.03 0114 S05 FVIII (con)/vWF Def 34.4 33.7 1.02 0.96 0.97 0.99 48.4 49.2 0.98 0.98 0.98 1.00 0114 S06 FVIII (con)/vWF Def 34.4 33.4 1.03 0.96 0.97 1.00 48.6 48.9 0.99 0.98 0.97 1.01 0114 S07 FIXDef (con) 34.7 34.7 1.00 0.97 1.00 0.97 41.1 50.5 0.95 0.97 1.00 0.97 0114 S08 FIXDef (con) 34.6 32.8 1.05 0.97 0.95 1.02 48.5 48.9 0.99 0.98 0.97 1.01 0114 S09 FXDef (con) 41.8 39.7 1.05 1.17 1.15 1.02 60.7 59.2 1.03 1.22 1.17 1.04 0114 S10 FXDef (con) 36.4 36.2 1.01 1.02 1.05 0.97 51.1 51.4 0.99 1.03 1.02 1.01 0114 S11 FXIDef (con) 34.6 34.4 1.01 0.97 0.99 0.97 48.8 49.7 0.98 0.98 0.99 1.00 0114 S12 FXIDef (con) 33.3 33.8 0.99 0.93 0.98 0.95 46.8 47.7 0.98 0.94 0.95 1.00 0114 S13 FXIIDef (con) 32.4 31.7 1.02 0.91 0.92 0.99 44.9 45.3 0.99 0.91 0.90 1.01 0114 S14 FXIIDef (acq) 34.4 35.5 0.97 0.96 1.03 0.94 49.4 53.4 0.93 1.00 1.06 0.94 0114 S15 LA Postive 71.2 34.1 2.09 1.99 0.99 2.02 121.8 49.7 2.45 2.46 0.99 2.49 0114 S16 LA Postive 80.8 35.5 2.28 2.26 1.03 2.20 137.4 51.7 2.66 2.77 1.03 2.70 0114 S17 LA Postive 65.3 34.4 1.90 1.82 0.99 1.83 107.3 49.8 2.15 2.16 0.99 2.19

[0069] As shown in Table 2, the normalized ratio reported for LA positive samples in the presence of 350 nM PPACK was not lower than the corresponding results from HemosIL® dRVVT (see Table 1), which indicates that the methodology in a mixing study setting in the presence of 350 nM PPACK has better sensitivity than the HemosIL® dRVVT assay.

[0070] As discussed below, while the PPACK model demonstrates a significant improvement in the current LA testing methodology, the uniqueness of various thrombin and factor Xa inhibitors provides an even richer inventory of arsenal in tackling the LA test sensitivity and specificity, with the use of different inhibitor combinations.

[0071] Referring to FIG. 7, I-2581, a synthetic thrombin inhibitor, decreases the dRVVT screen-to-confirm ratio for both LA positive and NPP samples when used in the dRVVT assay, which could potentially further increase the LA assay specificity when used together with an inhibitor with the capability of increasing the LA assay sensitivity.

[0072] In yet another embodiment, referring to FIG. 8, the combined use of hirudin and argatroban, two direct thrombin inhibitors, provides another example of the power of such combinations in relation to the heterogeneity of LA antibody target antigens. Hirudin increased the dRVVT Screen-to-Confirm Ratio for both LA positive and NPP samples (left panel, FIG. 8). Although the use of hirudin in the dRVVT test increased the normalized ratio of the LA positive sample as shown in FIG. 9, it could also easily misclassify normal samples as weak positive without normalization (FIG. 8). The use of argatroban showed limited increase in NR for LA positive samples (FIG. 9) by decreasing the Screen-to-Confirm Ratio for NPP (FIG. 8). Hirudin and argatroban, when used together in the mixing study, increased the LA test sensitivity (NR: 1.66 at 200 ng/ml hirudin and 500 ng/ml argatroban as shown in FIG. 9), as compared to results for the HemosIL® dRVVT assay (NR: 1.64, data not shown), while the potential misclassification for normal samples is minimized (FIG. 8).

[0073] In another aspect, the invention is a system for aiding in the diagnosis of APS, comprising a clinical analyzer, e.g., but not limited to the ACL TOP® Family of clinical analyzers (Instrumentation Laboratory Company, Bedford, Mass.), configured to (a) bring a plasma sample in contact with a first phospholipid-dependent clotting assay reagent comprising a low concentration of phospholipid and a coagulation inhibitor for a sufficient amount of time to determine a first clotting time, (b) a second phospholipid-dependent clotting assay reagent comprising a high concentration of phospholipid and a coagulation inhibitor for a sufficient amount of time to determine a second clotting time, (c) a computing device having a processor in operable communication with said analyzer unit, and, (d) a non-transient machine readable media including instructions executable by the processor, the instructions when executed transform the first and second clotting times into a ratio, compare the ratio to a cutoff ratio, and establish an aid for diagnosing lupus anticoagulants based on the result of said comparison to said cutoff ratio.

[0074] Accordingly, the invention described here provides a new methodology in detecting LA. The usage of thrombin inhibitor PPACK on the detection of LA using the APTT and dRVVT assays is served as a model for the methodology in this invention. This invention leads to improved LA assay sensitivity/specificity and better utilization of mixing studies. Various thrombin inhibitors and factor Xa inhibitors, or a combination of such inhibitors, can be used to replace PPACK. Specifically, the current invention demonstrates that the synergistic effect from different coagulation inhibitors may be used to optimize the LA test sensitivity and specificity in relation to the heterogeneity of LA antibody target antigens.