Comparative analysis of Quanta Lite and Euroimmun enzyme-linked immunosorbent assays for the determination of antiphospholipid antibodies

INTRODUCTION

To make the diagnosis of antiphospholipid syndrome (APS) according to the updated official recommendations, a laboratory criterion is of crucial importance in addition to the clinical criterion.^[1,2] The laboratory criterion implies the presence of positivity of at least one of the three antiphospholipid antibodies (aPLs) (i.e., lupus anticoagulant [LAC], anti-cardiolipin [aCL] immunoglobulin (Ig) G/IgM antibody, and anti-β2-glycoprotein I [aβ2GPI] IgG/IgM antibody).^[1] Antibody positivity is observed at the level of the clinically significant cutoff value recommended by the manufacturer, namely, at moderate-to-high titers of aCL or aβ2GPI IgG or IgM (99^th percentile based on a reference population).^[2] The new 2023 APS classification criteria developed by the American College of Rheumatology (ACR) and the European Alliance of Associations for Rheumatology (EULAR) emphasize the importance of quantifying single-, double-, and triple-aPL positivity, distinguishing between IgG and IgM isotypes, and delineating moderate and high levels of aCL/aβ2GPI positivity.^[3] However, a major challenge for the laboratory is the consistency of positivity between tests from different manufacturers so that the laboratory criterion is reliable and has significant and real clinical value.^[4]

Among the laboratory tests used to assist in the diagnosis of APS, the determination of LAC has traditionally been based on a coagulation-based functional test that can be performed daily in the laboratory.^[5-9] However, there have been some concerns about the reliability of the routinely used three sequential tests: Screening, mix, and confirmation.^[10] For the determination of aCL and aβ2GPI, the most widely used tests are the enzyme-linked immunosorbent assay (ELISA).^[6] Basically, APS is a lifelong autoimmune disease, and the detected aPLs are heterogeneous in nature and can be detected in different clinical situations (thrombosis, unexplained miscarriages, conditions associated with eclampsia, and even in the elderly).^[6,7] The specificity of the antibodies used for detection is a particularly important methodological aspect that should be considered when discussing laboratory criteria. In addition to the initial determination of antibodies, it is recommended to perform at least a second measurement after 12 weeks, using the same test, method, and measurement principle.^[11] Although there are automated methods for the determination of aCL and aβ2GPI, most laboratories are only able to determine these antibodies using the ELISA method.^[12-14] In addition, some laboratories face the challenge of continuously purchasing ELISA tests from the same manufacturer over an extended period of time, which is necessary for stable patient monitoring. In addition to classification and diagnosis, reliable determination of aPLs is necessary for the decision on the use of anticoagulant therapy.^[15] Although great efforts have been made in establishing laboratory criteria for APS, research is still needed in the areas of harmonization and standardization, reproducibility, analytical specificity, and sensitivity of laboratory tests used to determine aPLs, and interpretation of results.^[16-20]

The main objective was to investigate the degree of agreement in antibody positivity between Quanta Lite and Euroimmun ELISA assays for the determination of aPLs due to their prevalence. We evaluated and compared the number of positive results of IgG and IgM aCL and aβ2GPI measured in patients with APS. In addition, we compared the laboratory calculated 99^th percentile and the manufacturer’s cutoff values using Quanta Lite commercial ELISA kits.

MATERIALS & METHODS

This study was conducted as an observational and cross-sectional study.

Subjects

Patients with primary APS (PAPS): 25.3% were men and 74.7% were women, out of a total of 79 patients; patients with APS associated to systemic lupus erythematosus (secondary APS-SAPS): 6.4% were men and 93.6% were women out of a total of 47 patients and 56 healthy individuals were selected for this study, of whom 28.6% were men and 71.4% were women. The average age of the patients was as follows: 49 years for men and 45 years for women in the PASP group, 70 years for men and 54 years for women in the secondary APS-SAPS group, and 46 years for men and 50 years for women in the healthy group. The diagnosis of APS was made on the 2006 revised Sydney criteria.^[1,2] Fetal loss was found in 50% (63 patients), thrombosis in 65% (82 patients), and both conditions were present in 15.9% (20 patients) of all examined patients (n = 126). All subjects in the healthy group were asymptomatic and showed no clinical signs of thrombosis, pregnancy morbidity, tumors, current infections, autoimmune diseases, and inflammation [Figure 1].

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Figure 1:

Flow diagram of subject recruitment.

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Samples were collected from January 2022 till August 2024, by a standardized venipuncture procedure^[21] and stored at −80°C until serial determination. Serum samples were collected in BD Vacutainer^® SST™ Tubes for the determination of aCLs and aβ2GPI, and plasma samples were collected in BD Vacutainer^® Citrate Tubes for the determination of LAC. Blood sampling was performed at least 3 months after the acute episode of clinical manifestation of APS. Each sample was determined in duplicate and the manufacturer’s procedure was fully followed.

This study was conducted with the approval of the Ethics Committee of the Clinical Hospital Center Bežanijska kosa, Belgrade, Serbia, number 2581/1 of April 12, 2011.

RESULTS

We found that there was a significant difference between the manufacturer’s and calculated cutoff values for aβ2GPI IgG and IgM, in all 126 patients with APS. Using Quanta Lite ELISA kit, we found lower cutoff values for aβ2GPI IgG and IgM. The calculated cutoff value (95% confidence interval [CI]) for aβ2GPI IgG was 10 SGU (9.46–10.54) (manufacturer’s value is 20 SGU), and for aβ2GPI IgM was 10 SMU (9.51–10.49) (manufacturer’s value is 20 SMU). Our study found that there was no difference between the manufacturer’s and calculated cutoff values for aCL IgG and IgM. The calculated cutoff value (95% CI) for aCL IgG was 14.5 GPL (13.76–15.24) (manufacturer’s value is 15 GPL), and for aCL IgM was 12 MPL (11.56–12.44) (manufacturer’s value is 12.5 MPL).

According to the laboratory criteria (manufacturer’s cutoff), 43 and 41 patients (both PAPS and secondary APS-SAPS) were classified as negative when using combined Quanta Lite and results of LAC and combined Euroimmun and results of LAC, respectively. Using Quanta Lite (manufacturer’s cutoff) and LAC, 42 patients were single positive, 26 patients were double positive, and 15 of them were triple positive. Using Quanta Lite (calculated cutoff) and LAC, 38 patients were single positive, 31 patients were double positive, and 20 of them were triple positive. Using Euroimmun (manufacturer’s cutoff) and LAC, 52 patients were single positive, 18 patients were double positive, and 15 of them were triple positive. The number of patients who did not meet the laboratory criteria when the calculated cutoff value was used instead of the manufacturer’s one decreased from 43 to 37 patients using Quanta Lite ELISA kit.

We investigated the frequency of positivity of the individual antibodies, which was determined with two different ELISA kits using the manufacturer’s cutoff values. The frequency of positive IgG aCL results was 8% (10/126) for the Euroimmun kit and 14% (18/126) for Quanta Lite. The frequency of positive IgM aCL results was 17.5% (22/126) for the Euroimmun kit and 35.7% (45/126) for Quanta Lite. The frequency of positive IgG aβ2GPI results was 7.1% (9/126) for the Euroimmun kit and 7.1% (9/126) for Quanta Lite. The frequency of positive IgM aβ2GPI results was 31% (39/126) for the Euroimmun kit and 12% (15/126) for Quanta Lite. When we used the calculated 99^th percentile as the cutoff for each antibody determined with the Quanta Lite ELISA kit, the results were as follows: 14% (18/126) aCL IgG, 40% (50/126) aCL IgM, 17.5% (22/126) aβ2GPI IgG, and 27% (34/126) aβ2GPI IgM antibody.

The cutoff values recommended by the commercial test manufacturers were used to classify the patient’s results (PAPS and secondary APS-SAPS) as positive or negative. The results of agreement between these two commercial kits were presented in Tables 1 and 2 for aCLs and aβ2GPIs, respectively.

In addition, the agreement of positivity is presented graphically [Figure 2a and b].

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Figure 2:

Graphically presented agreement between positive results determined using Quanta Lite and Euroimmun Elisa kits for (a) anti-cardiolipins, and (b) anti-β2-glycoproteins, based on the manufacturer’s cutoff values.

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Results show substantial agreement for aβ2GPI IgG and aCL IgG, moderate agreement for aCL IgM, and fair agreement for aβ2GPI IgM using the manufacturer’s cutoff values.

Findings indicate substantial agreement for aCL IgG and aβ2GPI IgG, moderate agreement for aβ2GPI IgM, and reasonable agreement for aCL IgM using calculated cutoff values.

We also investigated and compared the number of positive results of the ELISA kits examined. Our results indicate that the aCL IgG and IgM measured by Quanta Lite have a greater ability to detect positive results in this population than that measured by Euroimmun (13.5% vs. 7.9% and 35.7% vs. 17.5%, respectively). We obtained the same ability to detect positive results for aβ2GPI IgG measured with Quanta Lite and Euroimmun kits (7.1%). We found greater ability to detect positive results for aβ2GPI IgM measured with Euroimmun compared to Quanta Lite (31% vs. 11.9%).

As mentioned above, there was a difference between the calculated and the manufacturer’s cutoff value for aβ2GPI IgG and IgM measured with the Quanta Lite ELISA kits, which also means a difference in diagnostic sensitivity for these antibodies. We obtained better sensitivity when we used the calculated cutoff value for both aβ2GPI IgG and IgM antibodies.

DISCUSSION

Since the detection of aPLs is a large part of the overall diagnosis of APS, it is necessary that the tests used to detect aPLs have a high degree of diagnostic specificity and sensitivity to identify these patients in a timely manner and take appropriate clinical action. In addition, the assessment of aPLs can make an important contribution to the prognosis of the disease itself as well as to the risk assessment for the development of clinical complications associated with APS (thrombosis and obstetric complications). Despite the large number of studies performed and published guidelines for the determination of aPLs, the results of the determination are still inconsistent, whether comparing different detection methods or tests from different manufacturers using the same method (principle of determination).^{[15,19,20,24]} Some of the possible causes of this problem may be: Lack of standardization in the determination of the cutoff value, the calibration and quantification method, the choice of solid phase, the type and source of antigen, and other analytical problems. Similar study as ours was conducted by Žigon et al.^[25] in which they clearly emphasize the issue of aCL and aβ2GPI standardization, as well as the interpretation of laboratory measures for clinicians in establishing an APS diagnosis.

Numerous previous studies indicate poor agreement between different assays for the detection of aPLs, to which a number of factors contribute: depending on the choice of solid phase, the detected antibodies are either bound to cardiolipin or directly to aβ2GPI.^[26-28] Consistent with prior research, aCL IgM positivity shows poorer agreement between different tests compared to aCL IgG. Similarly, aβ2GPI IgG positivity showed better agreement between tests than aβ2GPI IgM. Furthermore, the choice of the cutoff value affects the stratification into positive and negative results. When the calculated cutoff specific to the population is used, the number of positive/negative results for aβ2GPI IgG and IgM differs significantly. For aCL IgG and IgM, no significant difference was found in the number of positive/ negative results depending on the used cutoff. This is in line with the findings of Favaloro and Silvestrini^[26] who among other influencing factors, which showed that the choice of cutoff value influences the classification of results as positive or negative and that only 50% of the results given can be considered reliable, but even this value should be taken with caution (10% is the chance that the result is false positive or false negative).

The new ACR/EULAR classification criteria guidelines provide a better understanding of patients with regard to the diagnosis and treatment of APS.^[29,30] According to the latest classification criteria, three points are required to fulfill the laboratory criteria for the diagnosis of APS. A positivity of the IgM isotype (aCL and aβ2GPI) is scored with one point and that is not enough for fulfilling the laboratory criterion. Moderate positivity of the IgG isotype (aCL and aβ2GPI) is scored with 4 points and a high positivity of one of the isotypes (aCL and aβ2GPI) is scored with 5 points. If both isotypes (aCL and aβ2GPI) are present in a high titer, they are scored 7 points.

In addition to the analytical properties, the diagnostic properties of the test are also essential for a correct diagnosis. These depend largely on the chosen cutoff value (recommended by the manufacturer or calculated in the laboratory) at which antibody positivity is detected. Previous studies have shown that there are differences in the diagnostic sensitivity and specificity of antibodies determined with different tests, which is also supported by results of this conducted study.^[6,25,30] In addition, it is very important to evaluate the timing of sampling as well as the need to redetermine the aPLs to confirm the results.

The authors of this paper are aware of the fact that there are studies that have addressed this issue. However, even 20 years after the first results related to the harmonization and standardization of these tests for the detection of aPLs in APS, there is still a need to test the ELISA reagent kits of new manufacturers under routine conditions and to confirm the analytical performance specification of the manufacturer’s recommended methods in practice.

CONCLUSION

According to the recommended ACR/EULAR criteria for the diagnosis of APS, the type of aPL antibody contributes significantly to the number of points in making the diagnosis. Information about the laboratory where the test was performed and the type of the test used, should be available for all specialists that manage patients with APS, especially since it is recommended that a confirmatory result have to be obtained within 12 weeks. Our results showed substantial to fair agreement in the positivity of the antibodies determined with different ELISA tests. We have also shown that if the calculated cutoff value is used instead of the manufacturer’s cutoff value the number of positive results increases significantly. The most important issue in the determination of aPLs is the choice of the cutoff value. These findings may contribute to improved laboratory harmonization and standardization.