Interference of lupus anticoagulant causing antiprothrombin and anti-beta-2-glycoprotein I antibodies on international normalized ratio measurements: comparative analysis of international normalized ratio methods

Abstract

Background: Life-long vitamin K antagonist (VKA) therapy is recommended as a standard of care in antiphospholipid syndrome (APS) patients with thrombosis. Concerns have been raised about the validity of international normalized ratio (INR) measurements in lupus anticoagulant (LA)-positive APS patients because LA may interfere with phospholipid-dependent coagulation tests and could elevate INR measurements.

Objectives: Here, we aimed to determine the interference of antigen-specific monoclonal and isolated patient antibodies with LA activity on INR measurements.

Methods: Pooled normal plasma and control plasma from patients on VKA (without LA) were incubated with monoclonal and isolated patient immunoglobulin G antiprothrombin and anti-beta-2-glycoprotein I antibodies that express LA activity. INR was determined before and after addition using 3 laboratory assays (Owren STA-Hepato Prest, Quick STA-NeoPTimal, and Quick STA-Neoplastine R) and 1 point-of-care test device (CoaguChek Pro II).

Results: Antiprothrombin and anti-beta-2-glycoprotein I antibodies with LA activity interfered with recombinant human thromboplastin reagents (Quick STA-Neoplastine R and CoaguChek Pro II), particularly when added to plasma of VKA-treated controls. This effect was most evident on point-of-care test INR measurements, while the recombinant Quick reagent exhibited a lesser degree of interference. In contrast, tissue-derived thromboplastin reagents (Owren STA-Hepato Prest and Quick STA-NeoPTimal) remained largely unaffected by these antibodies, both in pooled normal plasma and VKA anticoagulated control plasma. Among these reagents, the Owren INR reagent exhibited the lowest sensitivity to the influence of LA antibodies. This observed difference in sensitivity is independent of the plasma dilution factor or the presence of factor V or fibrinogen in Owren reagent.

Conclusion: INR reagents that utilize recombinant human thromboplastin are more sensitive to the presence of monoclonal and patient-derived antibodies with LA activity. Consequently, APS patients positive for LA should be monitored using tissue-derived thromboplastin reagents, given its reduced susceptibility to interference by LA-causing antibodies.

Keywords: anticoagulants; antiphospholipid syndrome; international normalized ratio; lupus coagulation inhibitor; warfarin.

Figure 1

Spearman correlation analysis to identify the correlation between the international normalized ratio (INR) assays. Whole blood point-of-care test (WB-POCT) vs STA-Hepato Prest (A), WB-POCT vs STA-NeoPTimal (B), WB-POCT vs STA-Neoplastin R (C), plasma-POCT vs STA-Hepato Prest (D), plasma-POCT vs STA-NeoPTimal (E), plasma-POCT vs STA-Neoplastin R (F), and WB-POCT vs plasma-POCT (G). The dashed 45° line represents the perfect concordance. P < .05 was considered statistically significant. r², Goodness of fit; r, Spearman correlation coefficient.

Figure 2

Bland–Altman analysis to identify the agreement between the international normalized ratio (INR) assays. Whole blood point-of-care test (WB-POCT) vs STA-Hepato Prest (A), WB-POCT vs STA-NeoPTimal (B), WB-POCT vs STA-Neoplastin R (C), plasma-POCT vs STA-Hepato Prest (D), plasma-POCT vs STA-NeoPTimal (E), plasma-POCT vs STA-Neoplastin R (F), and WB-POCT vs plasma-POCT (G). The solid line indicates mean differences, and the dotted lines represent the upper and lower limits of agreement (95% CI).

Figure 3

Monoclonal antiphospholipid antibodies with lupus anticoagulant activity interfere with international normalized ratio (INR) measurements. Pooled normal plasma (PNP; A) or pooled vitamin K antagonist-treated plasma with INR values at the low (A; ≈2.0), middle (B; ≈3.0), and upper (C; ≈4.0) parts of the therapeutic range were incubated with monoclonal antiprothrombin antibodies (28F4 and 3B1), monoclonal anti–beta-2-glycoprotein I antibodies (27G7 and 3B7), or both. INR measurements were performed using STA-Hepato Prest (I), STA-NeoPTimal (II), STA-Neoplastin R (III), or point-of-care test (POCT; IV) INR reagents. INR = limit of detection.

Figure 4

Patient-derived antiphospholipid antibodies with lupus anticoagulant activity interfere with international normalized ratio (INR) measurements. Pooled normal plasma (PNP; A) or pooled vitamin K antagonist-treated plasma with INR values at the low (A; ≈2.0), middle (B; ≈3.0), and upper (C; ≈4.0) parts of the therapeutic range were incubated with 0 to 400 μg/mL patient-derived antiprothrombin antibodies or anti–beta-2-glycoprotein I (aβ2GPI) antibodies. INR measurements were performed using STA-Hepato Prest (I), STA-NeoPTimal (II), STA-Neoplastin R (III), or point-of-care test (POCT; IV) INR reagents. INR ≈ limit of detection. aPT, antiprothrombin.

Figure 5

Interference of international normalized ratio (INR) values by lupus anticoagulants is independent of factor (F)V concentration in the INR reagents. Pooled normal plasma (PNP) was incubated with 200 μg/mL monoclonal anti–beta-2-glycoprotein I antibody (27G7; A–C) or antiprothrombin antibody (28F4; D–F) in the presence or absence of added FV (20 nM). Results are shown as mean ± SD (n = 3). INR measurements were performed using STA-Hepato Prest (A, E), STA-NeoPTimal (B, F), STA-Neoplastin R (C, G), or point-of-care test (POCT; D, H) INR reagents. Ns, not significant.

Figure 6

Interference of international normalized ratio (INR) values by lupus anticoagulants is independent of fibrinogen concentration in the INR reagents. Pooled normal plasma (PNP) was incubated with 200 μg/mL monoclonal anti–beta-2-glycoprotein I antibody (27G7; A–C) or antiprothrombin antibody (28F4; D–F) in the presence or absence of added fibrinogen (10 μM). Results are shown as mean ± SD (n = 3). INR measurements were performed using STA-Hepato Prest (A, E), STA-NeoPTimal (B, F), STA-Neoplastin R (C, G), or point-of-care test (POCT; D, H) INR reagents. Ns, not significant.