Lupus Anticoagulants/Phospholipid-Dependent-Antibodies

July 13, 2003
08:00 to 12:00
Hall 4
The International Convention Center, Birmingham

Chairman: Ph. G. De Groot, The Netherlands
Co-chairs: J. Arnout, Belgium; M. Galli, Italy;; S. Machin, UK; J.
H. Rand, USA; R. Roubey, USA

Number of attendees: about 300

Introduction: The chairman opened the meeting by reviewing the diagnostic criteria for the antiphospholipid syndrome. He reviewed the literature on the standardisation efforts performed and concluded that the assays used for the diagnosis are far from standardised. He stated that based on these studies that it is impossible to understand and to interpret the literature on antiphospholipid antibodies. The outcome of the assays to detect the presence of antiphospholipid antibodies is so dependent on the laboratory where the tests are performed that comparison of experiments from different laboratories is impossible. He gave some examples of possible variations in assay conditions that strongly influence the outcome of the assays. Finally he made an appeal on the audience to start working parties for the development of strict criteria for the assays

I.    Criteria for the diagnosis of APS

Dr Pengo (Italy) sent a questionnaire to 54 centres in Italy regarding the diagnosis of APS and obtained responses of 47 %. He also presented a number of cases to the audience and compared the answers got by hand rising with the answers got with the Italian questionnaire. In general there was a good agreement in the audience on the definition of whether the presented cases were APS or not and how to treat the proposed patients.
In the second part of his presentation Dr Pengo showed his results on the criteria for the diagnosis of APS and concluded that thrombotic complications are mainly seen when more that one serological tests for APS were positive, the highest score was seen when lupus anticoagulant, anticardiolipin antibodies and b2GPI antibodies were positive.

Dr Cohen (UK). The finding that lupus anticoagulant (LA) and/or anti-beta2-glycoprotein I (b2GPI) antibodies are more strongly associated with thrombosis than are anticardiolipin antibodies (aCL) has led to the proposal of a new classification  (Lupus Anticoagulants/Phospholipid-Dependent-Antibodies SSC, ISTH, Boston, 2002) for the Antiphospholipid Syndrome (APS). Under this proposed classification Type I patients are those positive for LA and anti-b2GPI antibodies; Type II those with LA alone; Type III those with anti-b2GPI alone and Type IV "all the rest". We have classified 123 patients with persistent antiphospholipid antibodies (aPL), attending UCL Hospital for at least 6 months, according to the ISTH SSC 2002 proposed serological criteria. The cohort was tested for LA, IgG and IgM anti-b2GPI, and IgG and IgM aCL. Ninety-six of these patients fulfilled Sapporo clinical criteria for APS and 70 had venous and/or arterial thrombosis. In both these clinical groups, patients with Type I aPL were found to have significantly higher levels of IgG aCL and anti-b2GPI than those with Types II, III and IV. However, 25.2% (31/123) of all the patients and 26.04% (25/96) of patients fulfilling Sapporo clinical criteria were positive for aCL only. Omission of aCL testing from clinical investigation for APS could lead to a failure to diagnose the syndrome in a proportion of patients.

The conclusions of Dr Cohen is completely opposite the conclusions of Dr Pengo and after a thorough discussion they concluded to exchange samples to verify whether the tests they are using give the same results

Dr Galli (Italy) reviewed a wide selection of clinical studies on antiphospholipid antibodies, and formed the opinion that they are, at best, inconclusive. Only lupus anticoagulants were consistently associated with thrombosis, which implies that measuring them is helpful to define the patients’ risk of arterial and venous thrombosis, and to guide therapeutic management. The results of studies on anticardiolipin and anti_2-glycoprotein I antibodies are less convincing and partly controversial, but they leave open the possibility that their measurement too may be practical and useful, at least in some situations. At present, there does not seem to be any role for measurement of antiprothrombin antibodies. We foresee the need for well-designed clinical trials, to help establish which, if any, among the various antibodies, are risk factors for the antiphospholipid syndrome. To accomplish this, standardisation or at least harmonisation of the methods used to detect the antiphospholipid antibodies is mandatory. Without it, any clinical study will be criticisable and unable to reach firm conclusions.

II.    Standardisation of assays to detect the antiphospholipid syndrome.

Dr Arnout (Belgium) discussed the preparation of an international LA reference plasma panel. He presented the results of a working party (Drs. Arnout, Mackie, Jennings and Grey). The panel will consist out of plasma derived samples (moderate/strong, weak/moderate and negative) and monoclonal antibody spiked plasmas ( anti-b2GPI, anti-prothrombin and a combination; strong, moderate, weak and negative) 5000 to 10000 ampoules will be prepared. Before the end of September the panel will be tested in 6 laboratories (the working party + the labs of Drs de Groot and de Moerloose). After this, the samples will be distributed to 15 selected laboratories. Approval will be asked from the SSC and the WHO. The goal is that the reference panel is ready for distribution within a year

Dr Reber (Switzerland) gave two presentations. In the first he compared 10 commercially available ELISA kits for the detection of anti-b2GPI antibodies (a co-operative study together with Drs Tincani and Sanmarco). All but three assays were very good in detecting positive and negative samples; however, there was a large discordance when weakly positive samples were compared. In his second talk he hypothesised that part of the diversity of anti-_2GPI assays could arise from microheterogeneity in _2GPI protein used as antigen. In the second round, we sent a vial of "Forum" _2GPI (Hyphen Biomed) purified without perchloric acid precipitation to the participating centres, as well as a set of 6 calibrators (Forum Calibrators), humanised anti-_2GPI monoclonal antibodies (gift of T Koike) and 14 samples. Centres (n=12) were asked to run in parallel plates coated with the Forum and the local _2GPI using their local protocol. In 8/12 centres, calibration curves obtained with Forum Calibrators showed negligible differences between Forum and local _2GPI for IgG and IgM isotypes. As expected, those obtained with MoAb dilutions were even more similar. In the four remaining centres, marked differences were observed. Using Forum _2GPI did not reduce notably the spreading of samples’ numerical values as compared to local _2GPI. In 8/12 centres, the cut-off values were similar with both _2GPI. The use of percentiles resulted in a narrowing of cut-off values as compared with the first round. For IgG, concordance in sample classification was 9/14, which rose to 11/14 when excluding 2 outliners’ centres. For IgM, the corresponding figures were 1/14 and 9/14, respectively. Upon careful examination of local protocols, no technical reason could be found to explain the differences observed. Nevertheless, the concordance in sample classification improved as compared to the first round. Although these results are encouraging, additional efforts are required to improve anti-_2GPI assay standardisation.

III     Improvement of serological assays to detect APS

Dr. de Laat purified IgG’s of 20 patients that were found positive in a IgG dependent anti-b2-GPI ELISA were used to characterise domain specificity of anti-b2-GPI antibodies. To test this specificity he used domain deletion mutants. When the deletion mutants were coated to a plate the IgGs mainly recognised domain I and to a lesser extent domain V, although all domains were recognised. To investigate the specificity further, we coated full-length b2-GPI to a Ni-coated ELISA plate. Twenty IgGs were pre-incubated with domain I or domain II-V to Sepharose beads. We found that domain I alone was able to completely prevent the binding of the IgGs to full-length b2-GPI. Domains II-V were able to partly prevent the binding of the IgGs. The peptides spanning positively charged regions of domain I or V and the scrambled form of these peptides were ordered, and binding of these peptides coated to a plate by of 4 IgGs was observed. All the peptides were recognised, no difference was found between peptides with the original sequence and their scrambled counterparts. Also a randomly chosen peptide containing many arginines was recognised by all IgGs tested. These results indicate positive charge to be more important than conformation. In conclusion, plasma purified IgG antibodies mainly bind positive charges on domain I and V of b2-GPI.

IV     Laboratory registries

Dr. Tipodi discussed an exchange of samples in Italy to analyse the potency to detect lupus anticoagulants. In general there was a good agreement between the participating labs that follow the SSC criteria for the detection of lupus anticoagulants. The labs that did not follow the criteria scored factor deficient plasmas or heparin spiked plasmas also positive. The importance of strict criteria for the detection of lupus anticoagulants was proven again.

V    Clinical registries

Dr. Moll (on behalf of Dr Roubey) presented the progress of the APSCORE study, the registry of APS patients in the USA. After one year 415 patients enrolled the study, about 5000 plasma and serum samples are stored and about 1300 buffy coats are stored for DNA analysis. 52% of the enrolled patients had thrombosis (venous 26 %, arterial 19%, and both 8%). APSCORE uses a web-based data entry/data management system. Investigators that wish to obtain additional information can contact Robert Roubey (APSCORE@med.unc.edu).

Concluding remarks.

The present activities of the subcommittee includes:
  1. The preparation of international reference plasma panel for lupus anticoagulants. (To be delivered next SSC meeting)
  2. An update of the SSC guidelines for LA testing (to be discussed next SSC meeting)
  3. Guidelines for the ELISAs to detect anticardiolipin and antib2GPI antibodies (working parties being set-up)
  4. Continuous discussion on new serological criteria to define APS