Lupus Anticoagulants/Phospholipid-Dependent-Antibodies

June 17, 2004
14:00 to 18:00
Cenacolo Room
Fondazione Giorgio Cini

Chairman:  Ph. G. De Groot, The Netherlands
Co-chairs:  J. Arnout, Belgium; M. Galli, Italy; ; S. Machin, UK; V. Pengo, Italy;

J. Rand, USA; R. Roubey, USA


Number of attendees: more than 100

Introduction: The chairman opened the meeting and summarized the goals of this meeting. Dr. Pengo distributed a questionnaire with questions regarding antiphospholipid testing in diagnostic laboratoria. The goal of this questionnaire is to reach standardization in testing and the questionnaires were collected at the end of the meeting.

 

Prospective in LA diagnosis

Dr Pengo (Italy) discussed his recent finding that by using different Ca2+-concentrations to activate coagulation it is possible to discriminate between a b2-Glycoprotein I (b2GPI )-dependent  lupus Anticoagulants (LAC)  and a prothrombin dependent LAC. A reduction in final calcium concentration, from  10mM to 5mM, increased coagulation times in both dilute Russell Viper Venom Time (dRVVT) and dilute Prothrombin Time (dPT) when plasmas of patients with anti-b2GPI antibodies were used. Instead, all LA-positive anti-b2GPI antibody negative patients showed decreased coagulation times from mean These results are confirmed by running dRVVT of normal plasma spiked with affinity purified IgG anti-b2GPI antibodies. Therefore, when a PL–dependent coagulation test is run twice, at different final calcium concentrations, clinically important anti-b2GPI LA can be identified.

 

Dr. Arnout (Belgium) have used his panel of monoclonal antibodies directed against b2GPI and prothrombin that exert LAactivity to confirm the effects of different Ca2+- concentrations on dRVVT and dPT. He confirmed the original observations of dr. Pengo but showed that the presence of a high level of a anti-prothrombin antibody mediated LAC masks the presence of a b2GPI dependent LAC. Also the presence of an anti-factor VIII, LMWH. OAC etc should be carefully evaluated before the test as proposed by dr. Pengo could be introduced.

 

Dr. de Groot (the Netherlands) discussed the assay they use to detect a b2GPI –dependent LAC. This assay is based on the use of cardiolipin as confirming agent in a PTT-LA coagulation assay. His group studied the clinical of the detection of b2-glycoprotein I-dependent LAC in a cohort of 198 patients with autoimmune diseases. Presence of b2-glycoprotein I dependent LAC was strongly associated with a history of thrombo-embolic complications, an Odds Ratio of 42.3 (95% confidence interval 194.3 - 9.9) was found. An increased frequency of thrombosis was not found in the 33 patients with LAC independent of anti-b2-glycoprotein I antibodies (Odds Ratio 1.6, 95% confidence interval 3.9 - 0.8). The use of a LAC assay with cardiolipin as confirming agent strongly improves the detection of patients at risk for thrombosis. This suggest that anti-b2-glycoprotein I antibodies with LAC activity are antibodies responsible for the thrombo-embolic complications in APS.

In a separate study he compared dr. Pengo’s assay with the assay they developed in their own lab. In general a good correlation was found, although some discrepancies were noticed., probably due to the presence of mixtures of antibody populations.

 

A multicentre study was launched in which the clinical relevance of a b2GPI-dependent LAC will be tested in a cohort of at least 500 patients. This study will be performed in collaboration with and supported by Stago R&D (France)

 

Standardisation of the LAC assay

 

Dr Arnout (Belgium) discussed the development of a new standard for LAC testing. The standard should contain a mixture of patient pooled plasmas, normal pooled plasma spiked with monoclonal antibodies and normal pooled plasma. To make the standard, 10 L of patient plasma is needed. He suggested to collect 100 mL of plasma from 100 patients with high titer LAC. Dr Arnout distributed a form with which participants could enlist donors for this pool. after a long discussion on ethics and selection of patients a protocol and time table was accepted. The plan is to have the standard ready for the next meeting in Sidney. A steering committee (Arnout, de Groot, Mackie, Jennings, Gray, Sie, de Moorloose & Pengo) will guarantee the quality and progress of the procedure.

 

Dr. Mackie (United Kingdom) assessed a new dilute prothrombin time diagnostic assay, ACTICLOTÒ dPTtestä and ACTICLOT dPTconfirm (AdPT, American Diagnostica Inc, USA) for lupus anticoagulant (LA) detection. The ACTICLOT dPTtestä is a lipidated recombinant human tissue factor-based clotting assay designed to screen plasmas for LA. The ACTICLOT dPTconfirm test utilizes a high phospholipid-containing reagent to confirm the presence of LA. The clotting times are compared using calculations similar to those employed for DRVVT methods. AdPT was positive patients, 23 oral anticoagulant and 17 new thrombophilia outpatients. Samples were considered LA positive if they complied with International criteria and were positive in at least 2/4 reference methods (APTT ratio using Actin FSL, two DRVVT methods employing either high phospholipid or platelet neutralization confirm reagents, and StaClot LA). Two different lots of AdPT reagent were tested on an ACL300R analyzer and gave similar results in normals and LA patients. The AdPT reagents were stable for at least 96hr and the within run cv for normal and LA positive samples was <5%. Cut-off values for LA were based on AdPT results from normal subjects. 19/23 APA group patients had LA by AdPT (median and range 2.5 and 1.1-5.7 for patient/normal ratio; 1.4 and 0.9-3.7 for test/confirm ratio). All 17 thrombophilia patients were LA negative and AdPT negative. Using the APA and thrombophilia groups, and definite LA positive and negative samples, AdPT showed 83% sensitivity and 100% specificity. When AdPT was coupled with any of the other LA tests, sensitivity varied from 96-100% and specificity 82-100%. When a sensitive APTT reagent was used for screening, with AdPT and a DRVVT as specific tests, 96% sensitivity and 100% specificity were achieved. If three tests were performed, using APTTr mainly as a screening test, specificity was 100%. In oral anticoagulant patients, AdPT exhibited similar problems to other reagents, suggesting that mixing tests should be performed. The results suggest that AdPT is highly specific and detects the majority of patients with LA, albeit a slightly different spectrum of LA antibodies than seen with DRVVT or APTTr. ACTICLOT dPT used in combination with two recognized LA reagents provided the best diagnostic sensitivity for LA in patient samples studied in 20 healthy normal subjects, 23 known antiphospholipid antibody (APA)

 

Dr. Jennings (United Kingdom) discussed the effect of pH and buffering on DRVVT results with different commercial Kits. Following the observation that the dRVVT results of normal pooled plasma of  NEQUAS- distribution in England showed substantial variation he found that the pH of the plasma strongly influenced the results of LAC testing. When the pH of normal plasma is above 8.0, a dRVVT becomes positive. As the pH of lyophilised plasma after reconstitution can be above 8.0, he advised to use a Hepes buffer in dRVVT-testing.

 

Prospective in ELISAs

 

Guido Reber (Switserland): At the Sapporo consensus meeting [6], anti-β2GPI antibodies (aβ2GPI) were not included in the list of biological criteria defining the antiphospholipid syndrome (APS). At the SSC held in Boston July 2002, a new classification was proposed, which included aβ2GPI either alone or in the presence of lupus anticoagulant. One of the issues raised by adding this test to the biological criteria was the standardisation of these assays. Since 1999, our group has performed three collaborative studies on this topic. One study addressed the performances of homemade assays and the last one looked for the agreement between commercial tests (both were presented at the SSC meeting held in Birmingham July 2003). In summary, the results of our studies showed that the agreement was good for high positive samples, fair to poor for medium positive and poor for low positive. For commercial kits, there was agreement in sample classification (positive or negative) between the ten commercial kits in 12/22 samples for IgG isotype and in 5/22 for IgM isotype. Analysis of the data prompted us to make some proposals to improve the agreement between assays.

 

1)      To perform duplicates

2)      Control group for the determination of the upper limit of the reference range (cut-off)

3)      Calculation of the cut-off value

 

Because the distribution of the values is not Gaussian, the cut-off has to be calculated by the method of percentiles and not by adding standard deviations to the mean value. When analysing the distribution of the values of the control group, it must be kept in mind that anti-β2GPI antibodies are found in 2 to 5% of healthy individuals, especially in individuals aged more than 60. Kits' manufacturers are asked to indicate the units values corresponding to the 95%, 97.5% and 99% of the distribution of the control group in addition to the cut-off value they recommend. To fulfil regulation authorities requirements, the proposed cut-off takes into account sensitivity and specificity data obtained from clinical studies.

 

4)      Humanized monoclonal antibodies as reference calibrators

 

Because there are still no worldwide-accepted anti-β2GPI calibrators, we propose to use humanized MAb dilutions as calibrators to allow comparisons between assay systems. Our group is aware that a MAb, because it is directed to one single epitope, cannot mirror the diversity of the subsets of patients' autoimmune polyclonal antibodies with different binding affinities, generally lower than MAb. MAb may not detect micro heterogeneities in β2GPI preparations because they possibly do not affect their binding, as would be the case for subsets of patients’ antibodies. But MAb are not affected by batch-to-batch variation because their affinity do not change notably with time. This ensures that the stability of assay systems can be checked over many years.

 

We hope that these proposals will improve the agreement between assay results. Thirteen different commercial firms have agreed to follow this proposal and to include the humanised monoclonal antibodies as standard in their tests.

 

Does a panel of aPL tests improve diagnostics?

 

Monica Galli (Italy): A systematic review of the published articles on the antiphospholipid syndrome showed lupus anticoagulants were a clear risk factor for thrombosis, irrespective of the site and type of thrombosis, the presence of systemic lupus erythematosus, and the methods used to detect them. Anticardiolipin and antib2-glycoprotein I antibodies were possible risk factors of thrombosis, at least in some selected situations, whereas the measurement of antiprothrombin antibodies was not helpful to define the patient’s risk of thrombosis. In most cases, these studies analyzed the relationship with thrombosis of each single antiphospholipid antibody, rather than the combination of different antibodies with each other and/or with the coagulation tests commonly used to detect lupus anticoagulants. Based on these premises, we retrospectively studied a group of 103 well established lupus anticoagulant-positive patients for whom laboratory data about anticardiolipin, antib2-glycoprotein I and antiprothrombin antibodies were available, in order to investigate whether laboratory patterns emerged for their association with thrombosis in the antiphospholipid syndrome. First, anticardiolipin, antib2-glycoprotein I, and antiprothrombin antibodies were analyzed separately. We observed the following statistically significant associations. No association with arterial thrombosis reached significance.

 

Second, the antibodies were combined in different patterns, which included from 2 to 5 laboratory variables. Matching the variables for the antibody isotype, 60 laboratory patterns were generated. Their analysis with respect to three different types of thrombosis made us available 180 different combinations. Overall, 22 associations reached significance:

 

Type of thrombosis                  N. of significant associations/Total N. of associations

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- Any                                                                          14/60

- Venous                                                                      8/60

- Arterial                                                                       0/60

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In all but 2 cases, anticardiolipin antibodies > 40 units (G or any isotype) were present in the laboratory patterns that reached significance. Antib2-glycoprotein I antibodies (G or any isotype) were present in 11 significant patterns, and antiprothrombin antibodies (M or any isotype) in 7 cases. KCT Ratio of 1:1 mixture > 1.5 was present in 7 patterns which were significantly associated with thrombosis, dRVVT Ratio of 1:1 mixture > 1.5 in other 7 cases, and both KCT and dRVVT Ratios > 1.5 in one case. Increasing the number of variables of the laboratory patterns did not increase the Odds Ratio towards thrombosis. Figures 1 and 2 show the OR with 95% CI of the laboratory patterns which reached statistical significance. 

 

Conclusions

The analysis of the antibody patterns of a large cohort of lupus anticoagulant-positive patients confirmed that the presence of medium to high titres of IgG anticardiolipin antibodies, either alone or in various combinations, increases their risk of thrombosis. Conversely, the role of the other laboratory variables, irrespective of whether they were considered separately or combined, is less clear.

 

The way the results of the various antiphospholipid antibodies are reported may partly explain these findings. Unlike anticardiolipin antibodies, antib2-glycoprotein I and antiprothrombin antibodies were expressed in a qualitative fashion, which does not allow establishing clinically relevant cut offs. With respect to coagulation tests, dRVVT and KCT ratios did not show any correlation with the patients’ history of thrombosis. This is apparently in contrast with our previous observation that the dRVVT profile rather than the KCT profile was associated with thrombosis in lupus anticoagulant-positive patients. However, we must underscore that the two studies had different designs, and that in the present study we considered the ratio of the 1:1 coagulation times of the mixture of patient’s with normal plasma, in order to rule possible interference of oral anticoagulant treatment.

 

Vittorio Pengo (Italy) performed a comarable study. Over a 6-year period, 618 consecutive patients (55% of whom with previous documented thromboembolic events) were referred to their clinic for Antiphospholipid antibody detection. LA was detected according to internationally accepted recommendations. Enzyme-Linked-Immunosorbent Assays (ELISA) detect aCL and Anti-human b2GPI antibodies. Patients’ records were reviewed for the presence of previous thromboembolic events or obstetrical complications and each patient received a physical examination. LA (Odds Ratio 2.9, Confidence Interval 1.1-7.6) and ab2GPI (Odds Ratio 2.3, Confidence Interval 0.9-5.8) but not aCL (Odds Ratio 0.7, Confidence Interval 0.3-1.6) as individual test positively are significantly associated to thromboembolic events. The rates of thromboembolic events or obstetric complications were 94% (34/36) in patients with a triple positivity, 58% (18/31) in those aCL-and ab2GPI- positive but LA- negative, 50% (4/8) in patients in which ab2GPI antibodies was the single positive assay, 35% (7/20) in patients in which medium-high titre aCL was the single positive test, while no one of 5 patients in which Lupus Anticoagulant was the single positive test has had previous thromboembolic events. When results were tested in a logistic regression analysis, only the complete positive antiphospholipid profile was independently associated with thromboembolic events (OR 14.8, CI 3.5-62.2). ACL and ab2GPI antibody positive but LA negative profile showed an association with thromboembolic events without reaching statistic significance. The mean level of IgG Anti-human b2-glycoprotein I antibodies was statistically higher in triple positive profile and might account for positive Lupus Anticoagulant. These results show that LA and IgG ab2GPI antibodies as individual positive test but specially the combined positivity of all three tests in a single patient may be considerable for identifying patients with antiphospholipid syndrome (APS).

 

Dr. Machin (United Kingdom) commented on these observations and showed that in the patient population of his hospital a substantially number of APS patients had only a positive anti cardiolipin.

After an intense discussion agreement on headlines became apparent but no agreement could be reached on details. It was proposed that the chairman and cochairs should make a proposal on aPl testing and that this proposal should be proposed at the next meeting in Sidney. Hopefully, in Sidnet an agreement of new criteria on aPL testing could be reached.

 

Relevance of other antibodies

 

Dr. Rand (USA): gave an overview on annexin A5 and its possible role in APS. He discussed an assay he developed in his lab that was based on the inhibition of a clotting test by annexin A5. In the presence of aPL, the inhibitory effect of annexin A5 becomes less. The ‘shortening’ of the Annexin A5 mediated prolongation of the clotting test correlated with the presence of aPl, although there is an overlap between controls and patients. More studies are needed before this assay can be introduced as a aPL specific test.

 

Dr. de Groot (the Netherlands) investigated together with dr. Mackie the prevalence of IgG or IgM annexin A5 (A5) antibodies. An ELISA was performed with purified recombinant A5 coated onto the plate in two different laboratories on patients with various autoimmune diseases, mainly the antiphospholipid syndrome (APS). In laboratory A, 2/36 patients were positive for anti-A5 IgG, both patients had APS and had complications during pregnancy. 6/36 patients had anti-A5 IgM antibodies, 4/6 patients had a history of pregnancy loss. There was no association between thrombosis and A5 antibodies. In laboratory B, 26/198 patients were positive for IgM A5 antibodies with an odds ratio of 2.7 (significant) for thrombosis and 1.3 (non-significant) for pregnancy loss. 53 patients were positive for IgG antibodies with an odds ratio of 1.5 (non-significant) for thrombosis and 2.2 (non-significant) for pregnancy loss. In this laboratory the A5 antigen levels were also measured and were non-significantly lower in patients with IgM and/or IgG anti-A5 (7.9 ± 0.7 ng/ml plasma) than in patients without anti-A5 (9.8 ± 0.9 ng/ml plasma).  In conclusion, the measurement of annexin A5 antibodies seems to have no additional value for the detection of patient at risk for pregnancy loss. The relation with a history of thrombosis needs further studies.

 

Dr. Mackie (United Kingdom) investigated together with dr. de root the prevalence of IgG or IgM antiprotein S antibodies. A prototype commercial ELISA method for protein S antibodies was assessed in two different laboratories. The assay used microplates coated with highly purified human protein S, with bovine serum albumin in the blocking and wash buffers. Peroxidase conjugated monoclonal anti-human IgG or IgM were used for detection and humanised murine monoclonal anti-protein S for calibration. Good agreement was obtained between duplicate wells and replicate calibrant dilution curves on the same plate. The optical density of calibrant varied between plates and was therefore used to normalise the results. Each laboratory established separate normal ranges using 36-40 healthy normal subjects each. Laboratory A studied 14 serial samples from a 20-year-old female with thrombotic necrosis of the extremities. Six of these samples were IgG anti-protein S (aPS) positive and the results correlated with protein S activity, antigen and neutralising activity. aPS disappeared with treatment and clinical improvement. Each laboratory studied separate samples from patients with SLE, PAPS, lupus-like disease, pregnancy complications, acquired inhibitors and controls. Patients were judged APA positive if they had positive results for lupus anticoagulant, anti-cardiolipin or anti-B2GPI assays. Laboratory A studied 118 such samples, of which 18/86 APA positive samples and 1/32 APA negative samples had aPS (14 IgG, 5 IgM). Laboratory B studied 198 samples, of which 64/128 APA positive samples had aPS (54 IgG, 3 IgM, 7 dual positive); and 17/70 APA negative samples had aPS (14 IgG, 1 IgM, 2 dual positive). Overall, 38% of APA positive patients had aPS and 18% of APA negative patients; the presence of aPS thus having a high association with other APA (FET, p=0.0002). There were no significant associations of aPS with pregnancy loss or thrombosis, although further, prospective studies are required.

 

Treatment of APS patients

 

Guido Finazzi (Italy) discussed the optimal intensity of oral anticoagulation for the prevention of recurrent thrombosis in patients with the antiphospholipid antibody syndrome is uncertain. Retrospective studies have suggested that only doses of warfarin adjusted to achieve an international normalized ratio (INR) of more than 3.0 are effective, whereas a recent randomised clinical trial comparing high (INR range 3.0 to 4.0) vs. moderate (INR 2.0 to 3.0) intensities of anticoagulation failed to confirm this assumption. They conducted a randomized trial in which patients with persistent lupus anticoagulant and/or moderate to high levels of anticardiolipin antibodies and previous thrombosis were given either high-intensity (INR range 3.0-4.5, target 3.5) or standard intensity (INR range 2.0-3.0, target 2.5) warfarin therapy. We sought to determine whether intensive anticoagulation was superior to standard treatment in preventing symptomatic recurrent thromboembolism without increasing the rate of bleeding complications. The WAPS trial was launched in the framework of the SSC Subcommittee on antiphospholipid antibodies/lupus anticoagulants and started in July 1997.  A total of 109 patients were recruited in the trial and followed for a median of 3.6 years. Mean INR during follow-up was 3.2 (SD 0.6) and 2.5 (SD 0.3) (p<0.0001) in the high- and standard-intensity groups respectively. Recurrent thrombosis was observed in 6 of 54 patients (11.1 percent) assigned to receive high-intensity warfarin and in 3 of 55 (5.5%) assigned to receive conventional treatment (hazard ratio for the high intensity group, 1.97; 95 percent confidence interval 0.49-7.89). Major and minor bleeding occurred in 15 patients (2 major) (27.8%) assigned to receive high-intensity warfarin and 8 (3 major) (14.6%) assigned to receive conventional treatment (hazard ratio 2.18; 95 percent confidence interval 0.92-5.15). High-intensity warfarin was not superior to standard treatment in preventing recurrent thrombosis in patients with the antiphospholipid syndrome and was associated with an apour results with those of a similar trial recently published support the recommendation that conventional thromboprophylaxis with warfarin targeted at INR 2.5 is usually appropriate for these patients.

 

The results of this meeting in Venice are:

 

1. A collaborative study will be started  on the value of testing b2Glycoprotein I specific LAC (de Groot / Woodhams). Results will be presented in Sidney.

2. A standard will be developed for LAC testing (Arnout) The first results will be presented in Sidney.

3. A proposal for better standardisation for the b2GPI ELISA has been discussed (Reber). 13 different commercial firms have already agreed to follow these rules and to include the humanised monoclonal antibodies as standard in their tests. The proposal will be published.

4. A consensus is in reach for new criteria for aPL testing. A proposal will be formulated and presented in Sidney (chair and co-chairs).

5. The European WAPS study is finished (Finazzi). It is concluded that it is not necessary to treat patients with APS with high intensity warfarin. These results will be published. It is recommendated to treat patients with warfarin targeted at an INR of 2.5. Further analysis of the observery arm of the study will follow.