• antiphospholipid antibodies
• rheumatoid arthritis

We read with interest the letter entitled “Antiphospholipid antibodies and RA: presence of β₂GP1 independent aCL” by Bonnet et al published in the Annals in March 2001.¹ We believe that the letter needs additional clarification owing to inconsistencies in the terminology, methodology of antiphospholipid antibody (aPL) detection, and determination of positive values.

The use of the term “anticardiolipin antibodies” was somewhat misleading. The term was introduced and abbreviated as “aCL”, a group of antibodies detected in many conditions, but the β₂ glycoprotein 1 (β₂GP1) dependence of the aCL was not defined, even though the authors focused on β₂GP1 independent aCL. It is generally agreed that the term aCL, if not stated otherwise, defines the antibodies detected by the classical aCL enzyme linked immunosorbent assay (ELISA),^2,3—that is, both β₂GP1 dependent and β₂GP1 independent antibodies.

There were some potential methodological errors in determining β₂GP1 independent aCL. It was shown that antibodies against β₂GP1 (anti-β₂GP1) from patients with the antiphospholipid syndrome (APS) have the ability to bind β₂GP1 in complexes with cardiolipin only if the β₂GP1 concentration in solution is high enough. The threshold concentration of β₂GP1 was found to be just about 2 μg/ml, because no binding of anti-β₂GP1 was seen when serum samples were diluted 1:200 or more.⁴ As the physiological concentration of β₂GP1 in human serum is approximately 200 μg/ml, the threshold binding concentration is reached at a serum dilution of 1:100. In the presence of a relatively high concentration of endogenous β₂GP1, the statement that antibodies detected by this method are exclusively β₂GP1 independent is unjustified, as the sera containing high titres of anti-β₂GP1 might have yielded positive results by the method described in the letter.

The definition of antibody units in the letter is not clear and using Harris's standards for β₂GP1 independent aCL is not appropriate. With the use of Harris's standards,⁵ the units should be abbreviated as GPL (for IgG) and MPL (for IgM) as previously defined.⁵ However, Harris's standards were designed for use in the classical aCL ELISA and were prepared by pooling serum samples from patients with APS. Therefore, they contain mainly, or predominantly, β₂GP1 dependent aCL. β₂GP1 independent aCL were not defined in those standards and they were not meant as standards for β₂GP1 independent assays.

The interpretation of anti-β₂GP1 ELISA as a method to detect β₂GP1 dependent aCL may not be valid in all cases. It was shown that not all anti-β₂GP1 binding β₂GP1 adsorbed on polystyrene high binding plates also recognise β₂GP1 associated with cardiolipin. We reported this binding pattern for anti-β₂GP1 in children with atopic dermatitis,⁶ and the same was shown also for some patients with autoimmune diseases, including APS.⁷

The method for purification of β₂GP1 was not described. Because the authors focused on patients with rheumatoid arthritis (RA), it should be ensured that immunoglobulins were specifically removed from the β₂GP1 preparation. If this purification step was not carried out, traces of immunoglobulins in the β₂GP1 preparation might have yielded positive results for sera containing high titres of rheumatoid factor (RF). In fact, all sera containing IgM anti-β₂GP1 also had RF and the authors already suspected that this might be due to non-specific binding involving RF.

The method for determining cut off values was not explained and the number of normal human sera (NHS) included in the study as negative controls was not given. From the data presented in the letter, one may conclude that the cut off values were arbitrarily set at 20 units both for IgG and IgM isotypes of β₂GP1 independent aCL and for anti-β₂GP1. We recently compared the sensitivity of anti-β₂GP1 ELISA and classical aCL ELISA. The results showed great differences between their sensitivities and therefore also between the cut off values calibrated by the same standards.⁸ In addition, the authors did not report the proportion of NHS positive for each assay and the values of positive samples compared with patients with RA. Instead, they just referred to one study,⁹ which is only one of the several published estimations of aPL in healthy subjects.

We would like to support our criticism by adding some data about aPL in our patients with RA. We randomly selected 53 serum samples from patients fulfilling the ARA criteria for RA and 53 NHS as negative controls. The samples were tested for anti-β₂GP1, β₂GP1 dependent aCL, and β₂GP1 independent aCL. The assays were calibrated with β₂GP1 dependent monoclonal aCL (IgG and IgM anti-β₂GP1 ELISA and β₂GP1 dependent aCL ELISA) and positive in-house standards (all IgA assays and β₂GP1 independent aCL). The cut off values for anti-β₂GP1 were set as described⁸ by calculating the mean + 2 SD of logarithms of absorbance values for NHS and the 95th centile value of 32 NHS for both β₂GP1 dependent and β₂GP1 independent aCL. For the anti-β₂GP1 determination, we used affinity purified β₂GP1 adsorbed on Costar high binding plates as previously described.⁸ The β₂GP1 preparation did not contain any immunoglobulins. β₂GP1 independent aCL were tested as described in the letter, but the sera were diluted 1:200. Serum samples were tested simultaneously for β₂GP1 dependent aCL on the same plate by adding β₂GP1 in parallel duplicate wells. The final concentration of β₂GP1 was 10 μg/ml. This experimental design enabled direct comparison of binding to cardiolipin coated wells in the presence and absence of β₂GP1. For the final determination of β₂GP1 dependent binding, the values obtained in wells without β₂GP1 were subtracted from the values measured in wells with added β₂GP1. The patients' histories were evaluated for the occurrence of arterial or venous thrombosis and recurrent fetal loss. Statistical analysis was performed with the χ² test where appropriate.

Table 1 presents the frequency of positive sera in each group (NHS, RA, RA-RF positive, and RA-RF negative). The frequency of increased anti-β₂GP1, β₂GP1 dependent aCL, and β₂GP1 independent aCL was higher in patients with RA than in controls, but the difference was significant only for anti-β₂GP1. There were no differences in the frequency of any type of antibodies between the RF positive and negative patients. One patient (a male, 66 years old) had a history of deep venous thrombosis and pulmonary embolism together with positive anti-β₂GP1 and β₂GP1 dependent aCL of IgA isotype. Interestingly, 5/11 RA sera which showed binding to β₂GP1 adsorbed on a high binding plate did not recognise β₂GP1 associated with cardiolipin, as already reported.^6,7 In contrast, 3/9 RA sera binding β₂GP1 complexed with cardiolipin did not recognise β₂GP1 adsorbed on the surface of high binding plates. This phenomenon probably reflects the heterogeneous nature of anti-β₂GP1 in RA, which may differ in fine specificity from anti-β₂GP1 in APS.

The sera from our patients with RA exhibited an even higher frequency of β₂GP1 independent aCL than that reported in the letter. As expected from reported data, the presence of β₂GP1 independent aCL was not associated with signs of APS in our patients. We also found that the addition of β₂GP1 (10 μg/ml) lowered the binding of β₂GP1 independent aCL by about 50%, most probably owing to the competition between β₂GP1 independent aCL and β₂GP1 for the same binding sites on cardiolipin.

In conclusion, patients with RA may have anti-β₂GP1 and β₂GP1 dependent aCL, which might be associated with the signs of APS. The importance of distinguishing β₂GP1 independent aCL has not been fully clarified. It seems that β₂GP1 independent aCL do not confer an increased risk for APS in RA.