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Keywords:

  • β-2 glycoprotein I;
  • calcium;
  • lupus anticoagulant;
  • thrombosis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Blood sampling
  6. Lupus anticoagulant (LA)
  7. Anti-cardiolipin antibody ELISA (aCL)
  8. Anti-β2GPI antibody ELISA (antiβ2GPI)
  9. Modified dRVVT
  10. Dilute prothrombin time (dPT)
  11. Affinity purification of IgG antiβ2GPI antibodies
  12. Patients
  13. Results
  14. Discussion
  15. References

Summary.  Lupus anticoagulants (LA) are immunoglobulins which inhibit phospholipid (PL)-dependent coagulation tests. LA are not specific, as they may reflect the presence of antibodies to human prothrombin, human β2-Glycoprotein I (β2GPI), an association of previous antibodies or other antibodies. Antibodies to human β2GPI act as in vitro anticoagulants by enhancing the binding of β2GPI to PL, and this binding may be influenced by calcium ion concentration. A reduction in final calcium concentration, from 10 mm to 5 mm, increased coagulation times in both dilute Russell Viper Venom Time (dRVVT) and dilute Prothrombin Time (dPT) when plasmas of patients with antiβ2GPI antibodies were used. Ten LA patients showed increased dRVVT and dPT ratios from means of 1.5 to 1.7 (P < 0.001) and 2.4 to 4.3 (P = 0.002), respectively. Instead, all LA-positive antiβ2GPI antibody-negative patients showed decreased coagulation times from mean ratios of 1.5 to 1.3 (P = 0.004) in dRVVT and from 2.0 to 1.5 (P = 0.01) in dPT. These results are confirmed by running dRVVT of normal plasma spiked with affinity purified IgG antiβ2GPI antibodies. Therefore, when a PL–dependent coagulation test is run twice, at different final calcium concentrations, antiβ2GPI LA can be identified.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Blood sampling
  6. Lupus anticoagulant (LA)
  7. Anti-cardiolipin antibody ELISA (aCL)
  8. Anti-β2GPI antibody ELISA (antiβ2GPI)
  9. Modified dRVVT
  10. Dilute prothrombin time (dPT)
  11. Affinity purification of IgG antiβ2GPI antibodies
  12. Patients
  13. Results
  14. Discussion
  15. References

Lupus anticoagulants (LA) are immunoglobulins which inhibit phospholipid (PL)-dependent coagulation tests in the absence of specific coagulation factor inhibition [1–3]. Among the family of antiphospholipid antibodies, those detected by LA show a close association with both venous and arterial thrombosis [4–6]. Nevertheless, LA is a non-specific test as it may result from the presence of both antibodies to human prothrombin [7] and human β2-Glycoprotein I (β2GPI) [8,9]. The latter class of antibodies is associated with thromboembolic events, whereas antiprothrombin antibodies show a less striking association [6,10–14]. Therefore, in the presence of LA, it is important to classify the prolongation of the PL-dependent coagulation test as the result of the presence of functionally active antihuman β2GPI antibodies. Although this can be achieved by demonstrating the LA activity of affinity-purified antihuman β2GPI antibodies, this method is time-consuming and therefore not applicable in routine laboratory work [15]. Alternatively, titration of antihuman β2GPI antibodies can be measured by ELISA, but this method is not well standardized.

One method of improving coagulation tests exploring LA is to render them very sensitive to the presence of antiβ2GPI antibodies. The mechanism underlying the LA activity of antiβ2GPI antibodies is related to their ability to enhance binding of β2GPI to PL, thus impeding binding/activation of clotting factors [16]. As β2GPI binding to anionic PL and enhanced β2GPI binding to PL in the presence of specific autoantibodies may be influenced by calcium and ion strength [17], we evaluated the effects of various calcium concentrations in common tests exploring the presence of LA in patients with functionally active antiβ2GPI antibodies, and were able to identify plasmas of patients with clinically important antihuman β2GPI LA.

Blood sampling

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Blood sampling
  6. Lupus anticoagulant (LA)
  7. Anti-cardiolipin antibody ELISA (aCL)
  8. Anti-β2GPI antibody ELISA (antiβ2GPI)
  9. Modified dRVVT
  10. Dilute prothrombin time (dPT)
  11. Affinity purification of IgG antiβ2GPI antibodies
  12. Patients
  13. Results
  14. Discussion
  15. References

Venous blood was collected 9 : 1 in 3.8% sodium citrate and centrifuged at 2000 × g for 15 min at 4 °C. Plasma was then filtered through 0.22 µm Bio-Disc filters and stored at −80 °C until use.

Lupus anticoagulant (LA)

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Blood sampling
  6. Lupus anticoagulant (LA)
  7. Anti-cardiolipin antibody ELISA (aCL)
  8. Anti-β2GPI antibody ELISA (antiβ2GPI)
  9. Modified dRVVT
  10. Dilute prothrombin time (dPT)
  11. Affinity purification of IgG antiβ2GPI antibodies
  12. Patients
  13. Results
  14. Discussion
  15. References

The LA activity of plasma samples was studied applying dilute Russell Viper Venom Time (dRVVT) and Kaolin clotting time (KCT). The dRVVT was counted by an automated coagulometer (MLA Electra 1400 C, Haemoliance, Milan, Italy) using dilute viper venom and confirm reagents (DVV test and DVV confirm test, American Diagnostica Inc., Greenwich, CT, USA). Final results are reported as ratios between the coagulation time of a 1 : 1 mixture of patient and control plasmas, divided by the control plasma clotting time.

The KCT test was performed on a mechanical fibrometer (Mechrolab Clot-timer Model 202 A, Heller Laboratories, Santa Rosa, CA, USA), using a 2% Kaolin suspension and a 30 mm CaCl2 solution. Briefly: 100 µL of sample plasmas and 50 µL of Kaolin reagent (Sigma Chemical CO, St Louis, MO, USA) were incubated at 37 °C for 3 min. Fifty µL of prewarmed 30 mm CaCl2 were then added and the coagulation time was recorded.

Both test results (dRVVT and KCT) were considered abnormal if the ratio was above 1.1 on the basis of means +2 SD values obtained in plasma samples from 20 healthy subjects.

Anti-β2GPI antibody ELISA (antiβ2GPI)

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Blood sampling
  6. Lupus anticoagulant (LA)
  7. Anti-cardiolipin antibody ELISA (aCL)
  8. Anti-β2GPI antibody ELISA (antiβ2GPI)
  9. Modified dRVVT
  10. Dilute prothrombin time (dPT)
  11. Affinity purification of IgG antiβ2GPI antibodies
  12. Patients
  13. Results
  14. Discussion
  15. References

Quantitation of antiβ2GPI antibodies was tested as described elsewhere [18].

Normal values were considered up to 16 and 8 home units for IgG and IgM isotypes, respectively, considering 100 IgG and 100 IgM home units those obtained in two strong positive reference plasmas.

Modified dRVVT

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Blood sampling
  6. Lupus anticoagulant (LA)
  7. Anti-cardiolipin antibody ELISA (aCL)
  8. Anti-β2GPI antibody ELISA (antiβ2GPI)
  9. Modified dRVVT
  10. Dilute prothrombin time (dPT)
  11. Affinity purification of IgG antiβ2GPI antibodies
  12. Patients
  13. Results
  14. Discussion
  15. References

This test was performed by means of a mechanical clot timer (Mechrolab) by incubating 50 µL of test plasma, 50 µL of Russell Viper Venom (HA21, Abbott, Murex, Wienbaden-Delkenheim, Germany; lyophilized venom was reconstituted as recommended by manufacturer with 1 mL saline and then diluted 1 : 100 with Tris buffer), and 50 µL of indiluted PL (Phospholipid Reagent 0026, Unicorn, Cabru, Milan, Italy; each vial contains 20–50 µg of bovine brain lyophilized PL reconstituted with 3.5 mL distilled water) for 30 s at 37 °C and starting coagulation by adding 50 µL of the appropriate concentration of calcium ions (10 mm or 5 mm calcium chloride final concentration). Coagulation times for normal plasma (obtained using means ± 2 SD) were 20–25 s. Results are reported as ratios between the coagulation time of a 1 : 1 mixture of patient and control plasmas, divided by the control plasma clotting time.

Dilute prothrombin time (dPT)

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Blood sampling
  6. Lupus anticoagulant (LA)
  7. Anti-cardiolipin antibody ELISA (aCL)
  8. Anti-β2GPI antibody ELISA (antiβ2GPI)
  9. Modified dRVVT
  10. Dilute prothrombin time (dPT)
  11. Affinity purification of IgG antiβ2GPI antibodies
  12. Patients
  13. Results
  14. Discussion
  15. References

Recombinant thromboplastin (Ortho Recombiplastin, Haemoliance, Milan, Italy) was reconstituted with 20 mL of saline buffer, and then diluted at 1 : 20 with calcium chloride to obtain concentrations of 10 mm and 5 mm in the final coagulation mixture. 200 µL of diluted recombinant thromboplastin was added to 100 µL of filtered plasma on a 96-well polystyrene immunoplate (MaxiSorp, Nunc, Denmark) and coagulation times were recorded manually at the time of visual clot formation. Based on plasma samples from 20 normal subjects, the normal range (means ± 2 SD) was 31–35 s. Inter-assay and intra-assay variation coefficients were 2% and 3%, respectively. Results are expressed as the ratio between the coagulation time of sample plasma and pooled normal plasma (normal ratio 0.9–1.1). Fifty microliters of LA-positive plasma and 50 µL of normal pooled plasma were used for mixing studies. Experiments were run within 1 h at room temperature.

Affinity purification of IgG antiβ2GPI antibodies

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Blood sampling
  6. Lupus anticoagulant (LA)
  7. Anti-cardiolipin antibody ELISA (aCL)
  8. Anti-β2GPI antibody ELISA (antiβ2GPI)
  9. Modified dRVVT
  10. Dilute prothrombin time (dPT)
  11. Affinity purification of IgG antiβ2GPI antibodies
  12. Patients
  13. Results
  14. Discussion
  15. References

Total IgG was isolated from plasma by means of protein A Sepharose affinity chromatography, dialyzed against PBS, and loaded onto a prepacked NHS-activated Superose column (Pharmacia, LKB Biotechnology, Uppsala, Sweden) coupled with purified human β2GPI. The column was eluted using glycine NaCl pH 2.8. The purity of the resulting antiβ2GPI antibody preparation was checked by SDS-PAGE, and protein concentration was calculated by optical density at 280 nm [15]. Purified IgG preparations were tested for LA activity using the following mixture in dRVVT: 50 µL of normal pooled filtered plasma, 100 µL of purified normal or patient IgG in Tris buffer, 50 µL of PL (Phospholipid Reagent, Unicorn, Cabru, Milan, Italy) and 50 µL of Russell Viper Venom. After incubation for 30 s at 37 °C, 50 µL of calcium chloride (10 mm and 5 mm final concentration) were added and coagulation time recorded.

Patients

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Blood sampling
  6. Lupus anticoagulant (LA)
  7. Anti-cardiolipin antibody ELISA (aCL)
  8. Anti-β2GPI antibody ELISA (antiβ2GPI)
  9. Modified dRVVT
  10. Dilute prothrombin time (dPT)
  11. Affinity purification of IgG antiβ2GPI antibodies
  12. Patients
  13. Results
  14. Discussion
  15. References

As shown in Table 1, all patients were positive for LA, and were subdivided if LA was associated to the presence of antiβ2GPI antibodies that was ascertained in all cases by ELISA and in five cases by also evaluating LA activity of affinity-purified antiβ2GPI antibodies [15].

Table 1.  Antiphospholipid profiles of study patients
PatientsdRVVT Ratio*KCT Ratio*ELISA aCL GPL/MPLELISA antiβ2GPI IgG/IgM (home units)
  • *

     Ratio between coagulation time of patient and normal pooled plasma.

  • Statistical analysis.

Group A
# 12.21.8344/4040/16
# 21.91.8120/1273/23
# 31.31.6126/853/7
# 42.02.239/14611/99
# 52.92.594/3103/3
# 61.62.056/120292/154
# 71.31.475/5200/10
# 82.52.0464/11148/7
# 91.62.138/10344/14
# 102.96.598/14139/18
Group B
# 12.03.319/58/2
# 21.34.515/122/4
# 31.32.23/23/2
# 41.52.217/45/1
# 51.35.23/42/3
# 61.61.913/213/3
# 71.21.78/15/2
# 81.21.24/92/2
# 92.21.311/92/6
# 102.72.213/129/8
Normal values= 1.1= 1.1< 21/< 13< 16/< 8

Group A  Ten patients (2 male, 8 female) with a mean age of 47 years at the time of blood collection tested positive for LA, aCL and antiβ2GPI antibodies. Four had venous thromboembolic disorders, two arterial episodes, one multiple miscarriages, and three combined clinical events. All patients were classified as having APS, which was primary in six and secondary in the remaining four patients.

Group B  Ten patients (2 male, 8 female) with a mean age of 53 years tested positive for LA, but negative for aCL and antiβ2GPI antibodies. Two had thromboembolic disease, four systemic lupus erythematosus, one a fetal loss, one autoimmune thyroiditis, and two other autoimmune disorders.

Differences in mean coagulation times at various calcium concentrations in the two groups of patients were assessed by Student's paired t-test. A P-value equal to or less than 0.05 (two-tailed) was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Blood sampling
  6. Lupus anticoagulant (LA)
  7. Anti-cardiolipin antibody ELISA (aCL)
  8. Anti-β2GPI antibody ELISA (antiβ2GPI)
  9. Modified dRVVT
  10. Dilute prothrombin time (dPT)
  11. Affinity purification of IgG antiβ2GPI antibodies
  12. Patients
  13. Results
  14. Discussion
  15. References

The effect of calcium concentration in the classic dRVVT test was evaluated starting from a final concentration of 10 mm, and then going upstream and downstream. A representative experiment is shown in Fig. 1. Coagulation times of both normal plasmas and those of patients with LA tended to rise with increasing calcium concentrations, up to 20 mm. A different pattern was observed by decreasing calcium concentrations from 10 mm to 5 mm: no change in the coagulation time of normal plasma was observed, whereas that of patients who were LA positive but negative for antiβ2GPI antibodies decreased by 8 s, and that using plasma from a patient with antiβ2GPI LA increased by 8 s.

image

Figure 1. Results of a representative experiment showing dilute Russell Venom Time (in seconds) obtained at various final calcium concentrations in plasmas from a normal subject (circle), a patient with LA not related to the presence of anti-β2-Glycoprotein-I antibodies (triangle), and a patient with anti-β2-Glycoprotein-I Lupus Anticoagulant (square).

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When patients' plasma was tested at 10 mm and 5 mm final calcium concentrations, all antiβ2GPI LA patients showed an increase in coagulation times (Fig. 2a) from mean ratios of 1.5 (95%CI, 1.3–1.7) to 1.7 (95%CI, 1.6–1.9), and this increase was statistically significant (P < 0.001). Instead, all LA positive antiβ2GPI antibody negative patients showed a decrease in coagulation times when calcium concentration was decreased to 5 mm from mean ratios of 1.5 (95%CI, 1.2–1.9) to 1.3 (95%CI, 1.1–1.5) (P = 0.004) (Fig. 2b).

image

Figure 2. Dilute Russell Viper Venom Ratio (dRVVT of a 1 : 1 mixture of patient and control plasma divided by that from control plasma) at 10 mm and 5 mm final calcium concentrations in (a) plasma of patients withanti-β2-Glycoprotein-I Lupus Anticoagulant, and (b) plasma of patients with Lupus Anticoagulant not associated with anti-β2-Glycoprotein-I antibodies.

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This behavior was then tested in dPT. Preliminary experiments in this assay confirmed the crucial role of calcium concentration. As shown in Fig. 3, the shortest dPT for normal plasma was obtained at a final calcium concentration of 10 mm (dPT ranging from 31 to 35 s in 20 healthy subjects). Slight variations in the dPT of normal plasma were obtained by increasing or decreasing calcium concentration.

image

Figure 3. Results of a representative experiment showing dilute Prothrombin Time (in seconds) obtained at various final calcium concentrations in plasma from a normal subject (circle), a patient with LA not related to the presence of anti-β2-Glycoprotein-I antibodies (triangle), and a patient with anti-β2-Glycoprotein-I Lupus Anticoagulant (square).

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The coagulation time of a normal plasma sample was 32 s at 10 mm and 5 mm CaCl2, but rapidly increased to 60 s when calcium concentration was further reduced to 2.5 mm final concentration (data not shown). By increasing the calcium concentration to above physiological values, the coagulation times of normal plasma were 40 and 54 s with final calcium concentrations increasing to 15 mm and 20 mm, respectively. These variations were higher in plasmas from patients with LA, particularly in those plasma samples positive for LA and functionally active antiβ2GPI antibodies.

A marked prolongation in coagulation time for LA and antiβ2GPI antibody positive plasma was evident when the calcium concentration was reduced from 10 mm to 5 mm (from 64 to170 s).

When patients' plasma was tested in the dPT assay, the reduction in final calcium concentrations from 10 mm to 5 mm resulted in an increase in mean clotting time ratios from 2.4 (95%CI, 1.8–2.9) to 4.3 (95%CI, 3.2–5.4) in the group of patients positive for antiβ2GPI antibodies (Fig. 4a) – a very significant difference (P = 0.002). When LA was not associated with antiβ2GPI antibodies (group B), the mean ratio measured at 10 mm calcium concentration in these patients was 2.0 (95%CI, 1.6–2.5), and this value decreased to 1.5 (95%CI, 1.3–1.7) when the test was repeated using a reduced calcium concentration (P = 0.01) (Fig. 4b).

image

Figure 4. Dilute Prothrombin Time ratio (dPT of patient's plasma divided by that obtained in control plasma) at 10 mm and 5 mm final calcium concentrations in (a) plasma of patients with anti-β2-Glycoprotein-I Lupus Anticoagulant, and (b) plasma of patients with Lupus Anticoagulant not associated with anti-β2-Glycoprotein-I antibodies. Dashed line refers to results obtained with normal plasma.

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Finally, we verified the effect of different calcium concentrations on coagulation times obtained by using affinity purified antiβ2GPI antibodies. As shown in Fig. 5, the dRVT obtained using normal pooled plasma spiked with affinity purified antiβ2GPI antibodies (120 µg mL−1) showed a marked increase from 52.6 s to 148.8 s when final calcium concentration was reduced from 10 mm to 5 mm. On the other hand, when normal IgG used at the same concentration were spiked with normal plasma showed a modest increase in dRVVT from 34.5 s to 47.5 s that is consistent with that obtained using buffer instead of normal IgG (dRVVT from 36 s to 51 s).

image

Figure 5. Dilute Russell Venom Time obtained using normal pooled plasma spiked with normal or affinity purified anti-β2-Glycoprotein-I antibodies (120 µg mL−1) and final calcium concentration of 10 mm and 5 mm, respectively.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Blood sampling
  6. Lupus anticoagulant (LA)
  7. Anti-cardiolipin antibody ELISA (aCL)
  8. Anti-β2GPI antibody ELISA (antiβ2GPI)
  9. Modified dRVVT
  10. Dilute prothrombin time (dPT)
  11. Affinity purification of IgG antiβ2GPI antibodies
  12. Patients
  13. Results
  14. Discussion
  15. References

LA is an important test in vascular medicine, since its detection on two occasions 6 weeks apart in a patient with venous or arterial thromboembolism allows us to diagnose antiphospholipid syndrome (APS), and this in turn determines a different approach to secondary prevention and treatment. As thrombosis is a common event, laboratory diagnosis of LA becomes crucial in identifying patients with APS: guidelines for LA diagnosis have been reported [19], but correlations among various tests and proper standardization are still lacking [9,20].

When medium-high titer β2-glycoprotein I-dependent anticardiolipin antibodies and LA are both present in the same patient's plasma, a complete positive antiphospholipid profile may render physicians more confident with the diagnosis of APS. However, APS diagnosis becomes problematic, due to the above-mentioned laboratory pitfalls, when LA is the only positive test among those used to study antiphospholipid antibodies. Moreover, if antihuman prothrombin antibodies are responsible for LA, they are poorly associated with thromboembolic events [21]. Therefore, the development of simple coagulation tests improving the clinical significance of positive LA is important [22].

In this study we developed a new two-step assay to distinguish anti-human β2GPI LA from other antibodies with LA activity, the significance of which is still unknown. This result was obtained by running dRVVT at the final calcium concentration of 10 mm, followed by the same test at a final reduced calcium concentration of 5 mm. The same results were obtained when another test – dPT – was run at varioius calcium concentrations. Specific antiβ2GPI antibodies appear to be responsible for this effect since a marked prolongation of coagulation times were obtained at lower calcium concentration using normal plasma spiked with affinity purified antibodies.

Thus, a lower calcium concentration caused a marked prolongation in the coagulation times of patients with LA compared with those in whom LA was not related to the presence of these antibodies. On one hand, this phenomenon may be related to increased deposition of β2GPI-antiβ2GPI complexes over phospholipids and/or reduced competition of coagulation factors for phospholipid binding. In fact, reduced calcium concentration may increase the affinity binding of β2GPI to phospholipid surfaces [17] and this behavior may be amplified in the presence of specific antibodies (antiβ2GPI antibodies). On the other hand, β2GPI binding to immobilized PL may be unaffected by calcium concentration [23] and weaker in comparison with plasma coagulation proteins [24]: therefore, reduced calcium concentration in the system may greatly impair coagulation factor adsorption on a PL surface partially covered by β2GPI.

The results from the present study help to differentiate two types of LA. This is important, since laboratory work-up for the diagnosis of antiphospholipid syndrome is sometimes restricted to LA determination. Immunological tests are generally less reliable laboratory risk factors for thromboembolic events, and may be carried out in laboratories different from those in which LA is performed.

In conclusion, this study shows that a simple two-stage coagulation test is able to distinguish between LA related or not related to the presence of anti-human β2-GPI antibodies. Further studies are needed to validate the clinical significance of this distinction.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Blood sampling
  6. Lupus anticoagulant (LA)
  7. Anti-cardiolipin antibody ELISA (aCL)
  8. Anti-β2GPI antibody ELISA (antiβ2GPI)
  9. Modified dRVVT
  10. Dilute prothrombin time (dPT)
  11. Affinity purification of IgG antiβ2GPI antibodies
  12. Patients
  13. Results
  14. Discussion
  15. References
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