Department of Rheumatology, University Medical Centre, Ljubljana, Slovenia
Correspondence to: B. Boi
, Vodnikova 62, SI-1000 Ljubljana, Slovenia. E-mail: borut.bozic{at}guest.arnes.si
SIR, We read with interest the article by Audrain et al [1]. Unfortunately, they missed the opportunity to make further steps in approaching the complexity of the standardization procedure.
The authors start with a misleading definition of antiphospholipid antibodies (aPL). They mention the heterogeneous nature of aPL and diseases frequently associated with aPL, but not with clinical manifestations of antiphospholipid syndrome (APS). We would like to emphasize that antibodies binding to the cofactors are predominantly associated with clinical manifestations of APS, whilst aPL not associated with APS mostly bind directly to phospholipids.
The classical anticardiolipin (aCL) enzyme-linked immunosorbent assay (ELISA) employing bovine serum as the source of antigen is well established [2]. In the past, many modifications of the aCL ELISA have been described. The different results obtained with various kits in the study by Audrian et al. might have been a consequence of the manufacturers modifications of the classical aCL ELISA.
We note the lack of comment on the quite low agreement among the kits results for aCL detection in the different groups of patients defined by the authors, which was most probably a consequence of significant aCL heterogeneity. The concordance among the kits results was greater in the group of patients with primary APS, indicating lower heterogeneity of aCL in this group. However, differences among the kits could not be clarified by antibody properties, as important details of the assays were not presented in the article.
Anti-ß2-glycoprotein 1 (anti-ß2GP1) was not introduced as a heterogeneous group of antibodies; however, substantial differences in epitopic specificity and other properties of anti-ß2GP1 have been reported [3]. We have presented evidence for a specific subset of anti-ß2GP1 not associated with APS and differing considerably from anti-ß2GP1 in APS [4]. The influence of many assay variables on test results has also been demonstrated [5].
We agree with the authors that the weak agreement among the kits may be explained partly by different cut-offs. The statistical calculation of a cut-off (percentiles, standard deviations, multiples of the mean) can also result in different cut-offs for a single set of data. The authors should have included in the evaluation of kits enough sera from healthy donors for an appropriate definition of the cut-off in all kits for the same sera and the same statistical method. Such a procedure would have given additional strength to the data presented in the article, revealing real differences in the cut-offs among the kits and pointing also to eventual discrepancies between their cut-offs and the manufacturers.
The authors state that they could not establish parametric correlations among the kits due to the heterogeneity of the reported results, and expressed the need for an international reference serum. Selecting one serum or a few reference sera from their own positive samples would make an experimental step forward. Setting the calibration curve based on dilutions of such sera should be done in each assay. Such a procedure would give a satisfactory approximation of a quantitative comparison of the kits. A monoclonal IgG anti-ß2GP1 (HCAL) has already been proposed as the calibrator in assays for detection of aCL and anti-ß2GP1 [6]. Several papers have been published by our group in which HCAL and monoclonal IgM anti-ß2GP1 (EY2C9) were employed as standards [5, 7, 8]. We also presented cut-off values for different age groups defined for aCL and anti-ß2GP1 on the basis of these monoclonal antibodies [9, 10]. With the widespread use of the same monoclonal antibodies in research work, we could solve one of the main problems in the standardization of aCL and anti-ß2GP1 ELISA. If the authors had performed their experiments with a negative control group and monoclonal antibodies, they could have expressed the analytical sensitivity of each kit exactly as the concentration of monoclonal antibodies giving the signal equivalent to the cut-off [9, 10].
We cannot agree with defining the results of the aCL ELISA only as positive when calculating operating characteristics (clinical specificity, clinical sensitivity, positive and negative predictive values), as the diagnostic meaning of aCL correlates with the antibody level. According to the Sapporo criteria, only medium or high positive values are diagnostic for APS. The authors included low-positive aCL sera in the group of positives, which were subsequently used for the calculation of the operating characteristics. The operating characteristics in groups III and IV should have been calculated on the basis of clinical manifestations and not the diagnosis of established APS.
Any cut-off value is a reflection of the analytical sensitivity and of decisions about clinically relevant results. The evaluation of kits on the basis of clinical sensitivity and clinical specificity, considered as positives according to the manufacturers cut-offs, is inappropriate and such figures should be presented only as illustrations. Clinical specificity and clinical sensitivity are two mutually dependent variables and comparison of the kits results would have been more appropriate if clinical specificity had been compared at the same clinical sensitivity level and vice versa.
The authors have declared no conflicts of interest.
References
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