Number of attendees: 250-350

1. Diagnosis of APS: which assays should we recommend?

    Dr. Galli presented data on the thrombotic risk associated with different antiphospholipid antibodies currently measured such as Lupus anticoagulants (LAs), anticardiolipin (aCL), anti-ß2-glycoprotein I (aß2-GPI), and anti-prothrombin (aPT) antibodies. Her data were based on a systematic review of the relevant literature, published from 1988 to 2000. For the risk analysis of LA and aCL a large number of studies were analyzed giving information on more than 4000 patients. The association of LAs with thrombosis was significant in all studies, irrespective of the site and type of thrombosis. The association between aCL and thrombosis was only significant for a small minority of studies. LA appeared more strongly associated with thrombosis than aCL. From this analysis, it may be concluded that the detection of LA helps to identify patients at risk of thrombosis whereas measurement of aCL antibodies is of little value. Analysis of the thrombotic risk associated with aß2-GPI and aPT was mainly based on retrospective data. aß2-GPI appeared to be a risk factor for DVT but not for arterial thrombosis. aß2-GPI showed to be more strongly associated with thrombosis than aCL. Also aPT seem to predict DVT. The clinical relevance of aß2-GPI and aPT needs to be confirmed by prospective studies based on better standardized assays.
    Dr. F. de Groot and Dr V. Pengo reviewed how APS is currently defined and came up with a proposal for a new classification. They prepared this discussion together via e-mail conversation. Dr de Groot who presented the first part, critically reviewed the currently accepted diagnostic criteria (the so-called Sapporo criteria. Wilson et al. Arthritis & Rheumatism, 42; 1999: 1309-1311) and showed how internationally recognized experts continuously violate these. Dr. Pengo added a series of arguments why the Sapporo criteria are not optimal and proposed alternative criteria that separate APS into different types based on the antigen that is measured in the test. This presentation did as hoped initiate discussion and was concluded by the initiation of working party that would further discuss this important issue via e-mail.  The table with the proposed criteria for APS is as follows:

    Proposed Laboratory Criteria for APS

Type I	anti-ß2-Glycoprotein I and LA positive
Type II	only LA positive
Type III	only anti-ß2-Glycoprotein I positive
Type IV	all the rest (anti-PE, anti-prothrombin etc)

    *Measured on two occasions, at least 6 weeks apart.  Well defined normal range; IgG, IgM and IgA included.
 

    Dr. Finn Wisloff compared LA results obtained with an aPTT, a PT and a RVVT using identical phospholipid composition, instrumentation and calculation of the results. He found that about 20% of LA positive plasmas give discordant results. The majority of these discordant samples were low positive. These data are in support of the SSC criteria on LA testing that at least 2 different assay systems should be used. In addition, a uniform way of expressing the results should be recommended.
 

2. Clinical registries

    Dr. Guido Finazzi gave an update on the WAPS trial which was initiated by prof. Barbui under the auspices of our subcommittee. The purpose of this prospective study is two-fold: 1) in a randomized arm, to assess the risk/benefit ratio of high-dose oral anticoagulation vs. conventional antithrombotic prophylaxis; 2) in an observational arm, to obtain information on the natural history of non-randomized patients. 454 patients have been enrolled so far: 112 in the randomized and 342 in the observational arm. Median INR values of patients in the "high-dose" group were continuously around 3.2 as compared to around 2.3 in the "conventional treatment" group. Baseline samples of plasma, serum and DNA have been collected from all patients and are available for biological studies. Study projects are welcome and should be submitted for approval to the Chairmen and Steering Committee of the study. There was some discussion whether the difference in INR between the high intensity patients and the low intensity patients would be sufficiently large to come to a meaningful conclusion.
    Dr. Roubey presented a new initiative to register patients with APS: the Antiphospholipid Syndrome Collaborative Registry (APSCORE). The goal of this NIH funded registry is to enroll 1500 patients with APS and 500 individuals with antiphospholipid antibodies but without clinical manifestations of the syndrome. APSCORE will utilize a web-based data entry/data management system. Serum, plasma, and genomic DNA will be stored at UNC. Enrollment will begin in the summer of 2001. Investigators wishing to obtain additional information can take contact with Robert A. S. Roubey, M.D. Principal Investigator, APSCORE via the followinng E-mailaddress: apscore@med.unc.edu
 
3. Antiphospholipid antibodies: standardization issues

    Dr Ian Mackie discussed the importance of the use locally derived reference ranges as well as a standardized calculation of the test results. He presented data generated from the UK NEQAS surveys where various commercial DRVVT kits as well as ëin-houseí methods are used. Laboratories showed extreme variability in reporting their normal reference ranges. The need for local, analyzer and reagent, specific normal reference ranges for DRVVT is clear. The current SSC guidelines make no particular recommendations on the limits of normality. Arbitrary and generic reference ranges should not be used. Test/normal clotting time ratios should be performed for quality control purposes and to allow comparisons between laboratories and methods. The use of different algorithms for interpretation of the data has led to confusion between laboratories and also requires standardization. More precise recommendations by the subcommittee on locally derived reference ranges and a standardized way of expressing the results is needed.
    Dr. Ian Jennings reported on two recent UK NEQAS proficiency testing exercises in which the performance of potential LA controls or reference materials were evaluated.
In the first exercise, three plasmas were distributed to 43 centers in the UK. These plasmas comprised the National Institute for Biological Standards and Control (NIBSC) 1^ST British Reference Plasma Panel for Lupus Anticoagulant, a sample prepared using plasma from positive LA donors and one LA negative plasma pool. The results from this survey made it clear that LA screening performance with this reference panel is sub-optimal. The second set of data came from a recently completed UK NEQAS exercise, in which a series of plasmas spiked with monoclonal antibodies to b 2GP1, prothrombin and a mixture of each antibody, were distributed together with a LA negative plasma and a LA positive patient plasma. The majority of the 277 centers agreed on the diagnosis for each sample but complete consensus was not achieved. The results were agreement with earlier studies by dr Arnout who provided the monoclonal antibodies for this survey. Multicentre studies with such materials may be useful to assess assay sensitivity over a range of antibody concentrations and may allow further standardization, particularly in the accurate detection of weak LA.
    Dr. Guido Reber reported on a multicenter evaluation of the ELISA for ab₂-GPI antibodies. This study was initiated by the European forum on APL and involved 21 centers who received 2 normal samples and 28 patient samples selected by 4 centers. The participants also received 6 prediluted calibrators which enabled them to report their results into "Forum Units". Home made techniques as well as commercial kits were used. Marked differences in positivity rates were observed, ranging from 50 to 93% for IgG and 13 to 70% for IgM. Agreement was only found with 37% of the samples for IgG and for 27% of the samples for IgM. This study demonstrates that both home made assays and commercial kits for ab₂-GPI are poorly standardized. Two points have to be addressed: First, an uniform way to determine the cut-off of positivity should be tentatively adopted. Second, the influence of b₂GPI purification method and batch-to-batch consistency on assay results has to be evaluated.
    Dr. Siobhan Donohoe reported on a multicenter study initiated by our subcommittee to standardize the aPT ELISA. Protocols for a calcium free aPT method employed at UCH and a calcium containing method used at Utrecht were distributed to 15 centers. These methods were to be run in parallel with the in-house method. Human prothrombin, kindly provided by Diagnostica Stago was supplied to all participants along with microtitre plates. A series of 17 samples were distributed (April-May 2001). By lack of a suitable standard all assays were performed against the in-house standard. A high degree of variability was observed between the % activity of in-house standards making it impossible to compare results directly. However, by expressing the results in SD above Normal (< geo mean +2SD) it was possible to observe some consensus. More than 90% agreement on positivity or negativity was obtained with respectively 11 and 13 of the samples for aPT IgG and aPT IgM as determined with the calcium free protocol. Significantly less agreement was found with the other methods. A better standardization of aPT assays may thus be achieved but a international reference preparation is needed.
 
4. Antiphospholipid antibodies and prethrombotic state: new developments.

    Dr Françoise Dignat-George developed a method to measure endothelial microparticles (EMP) in plasma and showed previously that such microparticles display procoagulant properties. EMP may be measured by flow cytometry after immunolabelling with a monoclonal antibody against a vb 3. Plasmas of APS patients had significantly higher levels of EMP than controls plasma. Elevated EMP levels were also found in other patient groups. She also showed that plasma from APS patients and other patients induces vesiculation of cultured endothelial cells. However, when the procoagulant activity of EMP was assessed using a clotting assay, only EMP released in response to APS plasma were highly procoagulant. Dr Dignat-George proposes that by disseminating procoagulant activities in the circulation, EMPs could contribute to the hypercoagulable state of APS patients.
 
5. Concluding remarks.

From several questions raised from the participants it became clear that people expect some more precise guidelines on how to diagnose aPS, which assays are needed, useful and ready for widespread distribution. There is also need for an update of the SSC guidelines for LA testing.

Questions or comments regarding LA diagnostic criteria as well as concerning the newly proposed criteria for APS can be addressed to Dr.de Groot, Dr. Pengo as well as to the chairman. A working party will come together during this conference in Paris and will further work on these requests via email conversations in order to prepare the program for the next meeting in Boston:  Ph.G.deGroot@lab.azu.nl, pengo@ux1.unipd.it, jef.arnout@med.kuleuven.ac.be