Comparison of different kits in the detection of autoantibodies to cardiolipin and beta2glycoprotein 1

Marie A. P. Audrain, Françoise Colonna, Florent Morio, Mohamed A. Hamidou1 and Jean-Yves Muller

CHU Hotel-Dieu, Immunology Laboratory, Nantes and 1CHU Hotel-Dieu, Internal Medicine, Nantes, France.

Correspondence to: Marie Audrain, CHU Hotel-Dieu, Immunology Laboratory, Nantes, France. E-mail: marie.audrain{at}chu-nantes.fr


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion and conclusion
 References
 
Objective. To estimate the performance characteristics of 10 commercial kits and one in-house kit for the detection and quantification of anticardiolipin (aCL) (six kits) and anti-ß2glycoprotein 1 (anti-ß2GP1) (five kits) antibodies, and to evaluate the degree of variability between these different kits.

Methods. We determined the presence of aCL and anti-ß2GP1 IgG and IgM antibodies in 67 sera from 62 patients and reviewed the data separately. Each serum sample was tested with six commercial aCL determination kits and with four commercial and one in-house anti-ß2GP1 determination kit. We then analysed the operating characteristics of each kit (sensitivity, specificity, positive and negative predictive values) and we analysed the absolute and 2x2 agreements.

Results. The 62 patients included had primary antiphospholipid syndrome (APS) in 10 cases, secondary APS for eight, systemic lupus (SLE) for 23 and other diagnoses for the remaining 21. Operating characteristics differed from one kit to another. Good agreement was found using sensitive aCL determination kit and specific anti-ß2GP1 determination kit. Agreement between kits was medium for IgG aCL. 2x2 concordance studies showed a group of three aCL kits which were quite homogenous and showed that all anti-ß2GP1 kits formed quite a homogenous group.

Conclusion. A high degree of variability still persists for aCL antibody determination posing the question of the qualification of commercial or in-house kits and the question of standardization of results. A better concordance is found for high positive results. Good agreement exists for anti-ß2GP1 kits. aCL determination is still needed and should be complemented by anti-ß2GP1 determination.

KEY WORDS: aCL, anti-ß2GP1, antiphospholipid antibodies.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion and conclusion
 References
 
Antiphospholipid (aPL) antibodies consist of a heterogeneous group of auto-antibodies which may include antibodies directed against cardiolipin (aCL) or other phospholipids, both detected by ELISA and also lupus anticoagulant (LAC) detected by clotting tests. These antibodies are associated with various clinical features: venous or arterial thrombosis, unexplained fetal losses, eclampsia-associated prematurity or recurrent abortions. The association of at least one clinical criterion with one biological criterion (aCL present in medium or high titre and/or LAC in two or more occasions at least 6 weeks apart) defines the AntiPhospholipid Syndrome (APS) [1]. APS can be primary or secondary when occurring in patients with an autoimmune disease, particularly systemic lupus erythematosus (SLE). aPL antibodies can also be found in other circumstances in which they are not associated with clinical APS criteria, such as infections, malignancies and sometimes in healthy subjects.

It is now known that aPL antibodies are not only directed against phospholipids but also against a complex of phospholipid and phospholipid-binding plasma proteins called cofactors such as ß2glycoprotein 1 (ß2GP1) [2, 3] or prothrombin [4]. Furthermore, antibodies directed at these cofactors can be detected in ELISA in the absence of phospholipids [5, 6].

Tests for quantification of aCL or anti-ß2GP1 antibodies are performed with in-house or with commercial kits, but major difficulties still remain and the lack of standardization persists, which renders the inter-laboratory comparison, and sometimes the results, hazardous. International workshops have been organized and calibrated standards and international units (GPL and MPL) have been defined. Nevertheless, there are still some differences in results [711]. Determination of aPL antibodies is, however, important for diagnosis and for preventive or curative therapy.

The aim of this study was to estimate the performance characteristics of 10 commercial kits and one in-house kit for the detection and quantification of aCL (six kits) and anti-ß2GP1 (five kits) antibodies and to evaluate the degree of variability between different kits.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion and conclusion
 References
 
Methods
Six companies were approached in order to participate in this study. They were informed that assays would be done in our laboratory according to their instructions with sera from our lab. They were also advised that data would be published as an evaluation of ELISA kits for detection of aCL and anti-ß2GP1 antibodies without divulgence of the specific performance of any individual manufacturer and that they would be informed of their own identities in the numbering system. In our results, aCL kits are numbered 1–6 and anti-ß2GP1 kits are labelled A–E. The participating companies were (in alphabetical order): Biorad (Marne-la-Coquette, France), BMD (Marne-la-Vallée, France), Diasorin (Antony, France), Menarini (Antony, France), Pharmacia & Upjohn (St-Quentin en Yvelines, France) and The Binding Site (Saint-Egrève, France). All manufacturers gave quantitative results and cut-offs for negativity or positivity. These cut-off values were used for establishing the performance characteristics of each kit. Some kits reported results as ‘equivocal’ or ‘indeterminate’. In our analysis, these results were considered negative, since in Sapporo criteria only medium or high positivity was considered of diagnostic value. One in-house ELISA method was used for anti-ß2GP1 determination using ß2GP1 from Diagnostica Stago (Asnières, France) as described [12]. The validation of this assay was made using sera proposed for the European APL Forum ß2GP1-ELISA standardization [13].

LAC was determined in the haematology department according to the guidelines of the International Society on Thrombosis and Haemostasis, subcommittee for the standardization of lupus anticoagulants [14].

Sera
We selected 67 sera from 62 patients sent to the laboratory for routine aCL determination; a diagnosis was available for all of them. Sera were collected by venipuncture and stored at -20°C in aliquots until use in order to avoid freezing–defreezing cycles. Medical records of the studied subjects were retrospectively reviewed by one physician (hospital referent for systemic disease) without knowledge of the biological data. The clinical criteria for primary APS (PAPS) diagnosis were as follows [1]: vascular thrombosis (venous or arterial) or pregnancy morbidity (one or more unexplained fetal deaths beyond the 10th week of gestation; one or more premature births following severe pre-eclampsia or severe placenta insufficiency; three or more unexplained consecutive spontaneous abortions before the 10th week of gestation). The clinical criteria for secondary APS (SAPS) diagnosis were established as the association of APS as well as the criteria for autoimmune disease according to the American Rheumatology Association criteria [15]. SLE diagnoses were established according to the American Rheumatology Association criteria [15].

Clinical and biological data were registered and diagnosis was made associating at least one clinical manifestation and one biological criterion. The biological criteria were the presence of LAC or aCL antibodies on two or more occasions at least 6 weeks apart. aCL antibodies were determined with the aCL kit used for routine testing in our laboratory (kit 1). Anti-ß2GP1 were also determined for each serum but not taken into account for classification. The kit used for this determination was an in-house kit as previously described [12].

Patients were classified into four groups corresponding to diagnosis. Group I included 10 PAPS (11 sera), all positive for aCL and/or LAC. Group II included eight SAPS (nine sera), seven associated with SLE and one in a context of myositis, all positive for aCL and/or LAC. Group III included 23 SLE (24 sera), only one of whom showed clinical criteria of APS, although this patient did not have the biological criteria. It should be noticed that in this group, five women were under 25 and might not yet have shown episodes of thrombosis or obstetrical complications. Group IV included 21 other diseases (23 sera): clinical manifestations were thrombosis (six), recurrent fetal abortions (one), intra-uterine fetal growth retardation (one), coagulation disorders (four), medical history of thrombosis (three), renal graft (one), CREST syndrome (one), amyotrophic lateral sclerosis (one), cutaneous lesions (one), Horton (one) and autoimmune haemolytic anaemia (one).

Data analysis
For every kit, results were first reported as designed in the manufacturer's instructions. Quantitative results were considered negative, equivocal or indeterminate, weakly positive, positive or highly positive, depending on the manufacturer. Then, for determination of operating characteristics (sensitivity, specificity, positive predictive value and negative predictive value), we reported results as either negative or positive, using the cut-off values proposed by the manufacturers. It was decided to consider positive those that were called weakly positive, positive or highly positive and to consider negative those that were called negative, indeterminate or equivocal.

Sensitivity, specificity, positive predictive value and negative predictive value were defined as follows: sensitivity (Se), calculated as the frequency of a positive test in a population of patients with the disease, Se = TP/TP+FN; specificity (Sp), calculated as the frequency of a negative test in a control population, Sp = TN/TN+FP; positive predictive value (PPV), calculated as the frequency of patients with the disease in a population positive for the test as, PPV = TP/TP+FP; and negative predictive value (NPV), calculated as the frequency of disease-free subjects in a population negative for the test, NPV = TN/TN+FN. Where TP represents true positive, i.e. positive serum from a patient with APS defined as described above; TN represents true negative, i.e. negative serum from a patient without APS; FP represents false positive, i.e. positive serum from a patient without APS; and FN represents false negative, i.e. negative serum from a patient with APS.

Absolute concordance between kits was defined for IgG and IgM aCL and anti-ß2GP1 antibodies when all kits reported the same interpretation (negative or positive). Results of absolute agreement were expressed as number of sera where all kits agreed/number of sera tested with all kits. Concordance was also evaluated in 2x2 tables, and expressed as number of sera where two kits agreed/number of sera tested with these two kits.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion and conclusion
 References
 
We tested 67 sera with each aCL and anti-ß2GP1 kit. Each kit was tested in two different runs. For one aCL kit (kit 3), the second run was not validated because of low values on the calibration curve. We estimated that for this kit the number of sera available for analysis was too low. For one anti-ß2GP1 IgM kit (kit A), another problem appeared—almost all sera were positive. We estimated these positives as aberrant results. For these reasons, we decided to discard all results obtained with aCL kit 3 and anti-ß2GP1 kit A. In all tables we reported the number of sera used for the calculation.

Frequency of positive results
The frequency of positive IgG aCL results varied considerably from one kit to another: 18–64% (Table 1). The frequency of positive results in each of the four groups also showed great differences, more important in groups II, III and IV than in group I. Kits 1 and 6 showed 58 and 75%, respectively, of IgG aCL positive sera in group III, and 52 and 53%, respectively, of positive sera in group IV. However, kit 1 identified 82% IgG aCL positive sera in PAPS and 89% in SAPS. The other kits identified 55% of positive sera in PAPS and 44–50% in SAPS.


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TABLE 1. Frequency of positive results

 
Sensitivity, specificity, positive predictive value and negative predictive value
Results (based on the classifications TP, TN, FP and FN, as described above) showed relatively close sensitivity for IgG and IgM aCL (Table 2), and for IgG and IgM anti-ß2GP1 (Table 3). The sensitivity of the detection of aCL, either IgG or IgM, was between 65 and 90%. Specificities showed greater discrepancies for aCL determination, between 33 and 77%. In contrast, sensitivity of anti-ß2GP1 detection was very low and specificity was high. If another kit had been used to define the biological criteria, we would have obtained a similar classification except for one patient. This patient would move from group I to group IV. The reason was that LAC was present in almost all APS patients. Two patients from group IV with thrombosis but without aCL (with our reference kit) were aCL negative on all other kits except 1. In group III the one patient with clinical criteria of APS was aCL negative with all kits tested.


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TABLE 2. Sensitivity, specificity, positive predictive value and negative predictive value of aCL

 

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TABLE 3. Sensitivity, specificity, positive predictive value and negative predictive value of anti-ß2GP1

 
As in routine determination, we tested first for aCL antibodies, and then when aCL was positive, we tested for anti-ß2GP1 antibodies. We analysed sensitivity, specificity, positive and negative predictive values for anti-ß2GP1 antibodies in patients with aCL IgG or IgM positive results (Table 4). We observed that specificity and positive predictive value had increased, allowing us to find a good arrangement between sensitivity and specificity.


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TABLE 4. Comparison of sensitivity, specificity, positive predictive value, negative predictive value of anti-ß2GP1 in all patients versus aCL positive patients

 
Agreement within kits
We first reported quantitative results using the cut-off values proposed by the different manufacturers. Although using international standards for aCL determination, different manufacturers used different cut-off values and did not give the same interpretation of these quantitative values. Therefore, parametric correlations between values of aCL from different manufacturers were not possible as well as comparisons of the mean of positive and negative values. For anti-ß2GP1 antibodies, usually arbitrary units were used and no parametric correlation was possible. We chose to report results as qualitative results such as positive and negative using the specific cut-off of each manufacturer and we decided to consider indeterminate or equivocal results as negative and weakly positive as positive results. Then we analysed complete concordance between all kits as the number of sera where all kits agreed/number of sera tested and we analysed concordance between pairs of kits. Results showed that absolute agreement was medium for IgG aCL (51%) and correct for IgM aCL and IgG and IgM anti-ß2GP1 antibodies (Tables 5 and 6). We then tried to analyse absolute concordance in each group of patients (primary APS, secondary APS, SLE and other diseases). If considering IgG aCL in primary APS, the percentage of agreement was greater. In other groups, percentage of agreement was low. Such differences were not shown for IgM aCL and IgG or IgM anti-ß2GP1 antibodies. 2x2 agreement was expressed as the number of sera where two kits agreed/number of sera tested. For IgG aCL, results showed that kits 2, 4 and 5 could constitute a group quite homogenous with good concordance while kits 1 and 6 were different from each other. For anti-ß2GP1 antibodies, all kits formed quite a homogenous group.


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TABLE 5. Agreement between aCL kits

 

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TABLE 6. Agreement between anti-ß2GP1 kits

 

    Discussion and conclusion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion and conclusion
 References
 
Numerous commercial kits have become available for testing for aCL and anti-ß2GP1 antibodies. Several international workshops [810] have focused on the importance of standardization and several studies have been carried out to reach good standardization. In fact, for aCL antibodies this goal has not been fully achieved. All kits claimed to be standardized with the Louisville University standards. However, the cut-off levels of positivity proposed by each manufacturer were different. Two studies were conducted on only three kits and concluded that differences were observed [16, 17]. Then, the GEHT (Groupe d’Etude sur l’Hémostase et la Thrombose) conducted a study [18] with nine commercial kits and one in-house method defined by consensus between all the participants. This study concluded in a good agreement using the in-house method between participating centres. When using commercial kits, the percentage of positivity for IgG aCL ranged from 31.1 to 60%, and for IgM from 6.7 to 50%. The absolute concordance between kits occurred in 59% of sera for IgG and 51% of sera for IgM. Another study [19] was based on data from external quality assurance programs and concluded in a lack of consensus between laboratories. In two different programs, re-spectively 11.1 and 23.5% of total agreement was found for IgG and 20 and 47.1% of total agreement was found for IgM. This study emphasized the fact that the way to define the cut-off level point was critical. More recently, Reber et al. [20] conducted a collaborative study for standardization of the anti-ß2GP1 antibody assay in the framework of the European Forum on Antiphospholipid Antibodies Standardization Group. Twenty-one centres (15 using in-house methods and six using commercial kits) tested 30 serum samples. They concluded that in-house and commercial methods were poorly standardized especially in the lower range, whereas standardization was better in the medium and high ranges.

We planned our study in order to evaluate the agreement between several kits of aCL and anti-ß2GP1 antibodies and to approach the operating characteristics of each. As cut-off levels were not similar between kits, we decided to report results as positive or negative according to each manufacturer's instructions.

There was a medium agreement between all aCL kits especially for IgG isotype (51%). Three kits showed good agreement. In the PAPS group, the agreement was greater (64%). We could not focus on differences within each commercial kit manufacturer because all details of manufacturing were not available. However, it was clear that cut-off levels of positivity and interpretation of titres were different. The low level of agreement could be first explained by the fact that a lot of sera were weak positive for one manufacturer and would be ‘indeterminate’ or ‘equivocal’ for another, leading to a completely different interpretation: positive for one and negative for the other. Discrepancies were often observed with these low positive sera. A second explanation was that, with low positive values inter- and intra-assay reproducibilities were usually worse than with high values and the same serum testing in another run could be negative. A better agreement was observed in high positive values, emphasizing the fact that the choice of a commercial kit was critical for sera which were low positive or borderline.

Cut-off levels and interpretation were also different for anti-ß2GP1 antibodies, but the agreement between kits was better and even correct for IgG and IgM anti-ß2GP1 antibody determination considering all sera together or taking each group separately; this could be explained by the fact that the antigen was better characterized and no cofactor needed. In conclusion, a high degree of variability still persisted for aCL antibody determination kits and a good agreement was observed for the anti-ß2GP1 antibody kits.

With regards to sensitivity, specificity, positive and negative predictive values, results showed few differences from our previous study [12]. Several explanations could be made. First, in the previous study only one kit for aCL and one kit for anti-ß2GP1 antibody determination were tested. Secondly, this study was conducted with 191 consecutive sera with a lot of negative sera and 184 patients who were diagnosed as having other diseases, and only seven as having APS. However, the aim of the study was to focus on the role of anti-ß2GP1 antibody determination for diagnosis of APS. In this previous study, the number of APS patients was too low to calculate sensitivity. However, we could approach specificity. In the present study, the sera were not consecutive, but were included when having a positive aCL determination or a suspicion of APS (presence of aCL was confirmed on a second sample). The aim of this study was to compare several kits for aCL and anti-ß2GP1 antibody determination. We observed that the percentage of positivity for aCL IgG ranged from 18 to 64% for the six kits. For IgG and IgM anti-ß2GP1 antibodies the percentage of positivity ranged from 8 to 15% and 3 to 19%, respectively. We observed that three IgG aCL kits had low sensitivity, leading to a high specificity, one was highly sensitive and poorly specific and one poorly sensitive and poorly specific. A poor sensitivity was observed for aCL IgM determination. Sensitivity was, however, very questionable since the notion of a false negative was a matter of concern with some patients having APS without aCL but with a LAC or another aPL. In the international consensus [1], the other aPLs are not considered as a biological criterion, but we may not exclude formally a diagnosis of APS without testing all the aPL antibodies. In another way, specificity was also questionable since one cannot predict if a patient, who was aCL positive without clinical criteria, would not develop clinical APS later, especially if this patient was a young woman who could benefit from preventive treatment. Were they really FP sera since they were almost anti-ß2GP1 negative? Could we identify these patients as patients who would develop clinical APS later or could we identify them as patients who had true aCL antibodies without clinical criteria? Young patients included at the time of the study in groups III or IV might switch in the future and perhaps might be authentic APS without clinical criteria because of preventive therapy established as soon as aCL or LAC was detected. However, classification of patients according to aCL criteria, based on the other kits rather than our reference kit, did not show large discrepancies since only one patient switched from group I to group IV and possibly two from group IV to group I.

Anti-ß2GP1 antibody determination did not seem to be sensitive but was very specific. If we associated firstly a screening for aCL and a screening for anti-ß2GP1 antibodies in aCL positive patients, results in terms of sensitivity, specificity and predictive values were greater. We did not observe patients with isolated anti-ß2GP1 antibodies.

In conclusion, we observed that a high degree of variability still persists for aCL antibody determination, putting into question testing of sera by one or several kits. However, we observed that a better concordance is found for high positive results. This is in agreement with the Sapporo criteria for diagnosis of PAPS. Only medium or high values had to be considered as biological criteria. There were indications suggesting that a good agreement exists for anti-ß2GP1 antibodies.

As we could not establish parametric correlations, and as concordance was not perfect, the question of qualification and cut-off values of commercial or in-house kits is still being asked. Which sera could be used to participate in a reference panel in order to validate kits? High positive sera would favour specific but not sensitive kits, low positive sera would favour sensitive but not specific kits. Another question is the standardization of the different kits. They all used international standards and GPL and MPL units, but cut-off and results are different. The lack of standardization is easily demonstrated by national or international external quality programs. It would be interesting to define an international reference serum which could be included in each assay and which would allow us to recalculate result values using this reference, as is sometimes proposed.

Testing for anti-ß2GP1 antibodies in all aCL positive sera could help diagnosis and increase the specificity of a highly sensitive but low-specific aCL method. But, these anti-ß2GP1 antibodies are not yet considered as biological criteria, are of low sensitivity and a lot of patients had PAPS with aCL without anti-ß2GP1 antibodies.

We concluded that (i) aCL determination is still needed and could not be replaced by anti-ß2GP1 determination; (ii) it might be completed with anti-ß2GP1 determination and if all is negative with other aPL antibody determinations; (iii) as a lot of sera are borderline, some sera have to be tested on another kit; and (iv) a good working relationship between biologists and physicians is needed.

The authors have declared no conflicts of interest.


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion and conclusion
 References
 

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Submitted 28 June 2002; Accepted 1 August 2003