aCL Testing Research Articles

Standards and reference materials for the anticardiolipin and anti-β2glycoprotein I assays: A report of recommendations from the APL Task Force at the 13th International Congress on Antiphospholipid Antibodies

BackgroundThe confirmation of diagnosis of the Antiphospholipid Syndrome (APS) relies on laboratory tests. Current classification criteria for definite APS mandate the use of three “standardized” laboratory assays to detect antiphospholipid antibodies (aPL) [viz: anticardiolipin (aCL) IgG and IgM, anti-β2glycoprotein I (anti-β2GPI) antibodies IgG and IgM and/or a lupus anticoagulant (LAC)], when at least one of the two major clinical manifestations (thrombosis or pregnancy losses) are present. Several attempts have been made to standardize the aCL and anti-β2GPI tests, though, a considerable degree of inconsistencies still exist, limiting the clinical and diagnostic value of aPL tests. Among the areas of concern are the type and source of calibrant material, the lack of proper validated reference material and of universal units of measurement, particularly for anti-β2GPI antibodies. ContentA Task Force of scientists and leaders in the field from different countries — discussed and analyzed those critical questions in an evidence-based manner and further discussed and made recommendations at a workshop that was conducted during 13th International Congress on Antiphospholipid Antibodies (APLA 2010, April 13–16, 2010, Galveston, TX). SummaryThis concise report summarizes the findings, conclusions and recommendations of the task force and preconference workshop. The group recommended to ensure the availability of properly prepared and validated polyclonal and monoclonal antibody reference materials for both assays, to continue reporting the aCL assay in GPL/MPL units and to establish consensus international units of measurement for anti-β2GPI antibodies.

Docetaxel pharmacokinetics and its correlation with two in vivo probes for cytochrome P450 enzymes: the C14-erythromycin breath test and the antipyrine clearance test

Docetaxel has marked inter-patient PK variability, and metabolic phenotypic probes may enable individualised dosing. This is the first report directly comparing the erythromycin breath test (EBT) (a CYP3A4 probe) with the antipyrine clearance test (ACT), (a general CYP-P450/predominant CYP3A4 probe) for the correlation with docetaxel PK and toxicity. Patients pretherapy underwent: (A) EBT: IV C(14)[N-methyl]-erythromycin was administered and breath samples analysed for (14)CO(2), derived parameters included (1) (14)CO(2) flux at 10-min (CO(2)f(10)), (2) 20-min (CO(2)f(20)), (3) terminal rate constant k(CO2) and (4) AUC(CO2,(0-∞)) and AUC(CO2,(0-60).) (B) ACL test: patients were given oral antipyrine 10mg/kg, blood samples were taken for PK, and the clearance (CL(Ant)) was derived. Docetaxel was then given at 75mg/m(2)/3-weekly or 35mg/m(2)/weekly. Samples taken for docetaxel PK in first course on day 1 and PK parameters included clearance (CL(Doc)). Twenty patients accrued, docetaxel: 3-weekly/weekly=13:7. EBT parameters (N=19) (mean, [CV%]): CO(2)f(10) (%/min) 0.051 (106), CO(2)f(20) 0.052 (82), k(CO2) (min(-1)) 0.007 (22), AUC(CO2,(0-∞)) 7.9 (85), AUC(CO2,(0-60)) 2.64 (81). CL(Ant) (N=19) (ml/min); 35.8 (37). Docetaxel PK parameters (N=19): CL(Doc) (l/h)=57.2 (36), t(Doc1/2) (h)=12.7 (33). No correlations were observed between the docetaxel PK and EBT parameters. For docetaxel weekly patients, a significant linear relationship was observed between CL(Doc) and CL(Ant) (P=0.007, R (2)=79.47%). The utility of EBT for the prediction of docetaxel PK was not confirmed in this study. The antipyrine clearance test may be superior in this regard for docetaxel, but regimen dependent and hence warrants further evaluation.

IgM, but not IgA rheumatoid factor interferes with anti-cardiolipin and anti-β2 glycoprotein I measurements: a quantitative analysis

IgM rheumatoid factor (RF) is sometimes referred to as capable of causing interference in the IgM anti-cardiolipin (aCL) testing. Published guidelines are, however, inconsistent, and evidence regarding the interference is limited. Our goal was investigate IgM and IgA RF cross-reactivity and/or interference in IgM and IgA aCL and anti-β2 glycoprotein I (aβ2GPI) testing. Serum specimens with high IgM and IgA RF levels were tested for IgG, IgA and IgM aCL and aβ2GPI antibodies to examine cross-reactivity. Samples containing IgG aCL and aβ2GPI antibodies were spiked with IgM (and IgA) RF, and samples with high RF levels were spiked with IgG aCL antibodies. The mixtures were tested for IgM and IgA aCL and aβ2GPI antibodies. Specimens with high IgM and IgA RF concentrations did not test positive for IgM or IgA aCL and aβ2GPI antibodies (except one weak positive IgA aβ2GPI result), indicating the lack of cross-reactivity. In the spiked specimens, addition of IgM RF caused significant positive bias in the measurement of both aCL and aβ2GPI antibodies of IgM isotype in the presence of IgG aCL and aβ2GPI antibodies. The threshold for triggering significant interference was 318( )IU/ml for IgM RF, and 77 GPLU/ml for IgG aCL. Neither IgM, nor IgA RF, however, affected the IgA antiphospholipid (aPL) antibody testing. IgM RF can cause a false-positive IgM aCL result in the presence of IgG aCL antibodies. In studies on the prevalence and clinical significance of IgM aPL antibodies, RF interference should be considered and RF testing should be performed.

‘Criteria’ aPL tests: Report of a Task Force and preconference workshop at the 13th International Congress on Antiphospholipid Antibodies, Galveston, Texas, April 2010

Current classification criteria for definite antiphospholipid syndrome (APS) mandate the use of one or more of three positive 'standardized' laboratory assays to detect antiphospholipid antibodies (aPL) (viz: anticardiolipin [aCL] IgG and IgM; anti-β(2)glycoprotein I [anti-β(2)GPI] antibodies IgG and IgM; and/or a lupus anticoagulant [LAC]), when at least one of the two major clinical manifestations (thrombosis or pregnancy losses) are present. Although, efforts of standardization for these 'criteria' aPL tests have been conducted over the last 27 years, reports of inconsistencies, inter-assay and inter-laboratory variation in the results of aCL, LAC, and anti-β(2)GPI, and problems with the interpretation and the clinical value of the tests still exist, which affect the consistency of the diagnosis of APS. A Task Force of scientists and pioneers in the field from different countries, subdivided in three working groups, discussed and analyzed critical questions related to 'criteria' aPL tests in an evidence-based manner, during the 13(th) International Congress on Antiphospholipid Antibodies (APLA 2010, April 13-16, 2010, Galveston, TX). These included: review of the standardization and the need for international consensus protocol for aCL and anti-β(2)GPI tests; the use of monoclonal and/or polyclonal standards in the calibration curve of those tests; and the need for establishment of international units of measurement for anti-β(2)GPI tests. The group also reviewed the recently updated guidelines for LAC testing, and analyzed and discussed the possibility of stratification of 'criteria' aPL tests as risk factors for APS, as well as the clinical value of single positive vs. multiple aPL positivity. The group members presented, discussed, analyzed data, updated and re-defined those critical questions at a preconference workshop that was open to congress attendees. This report summarizes the findings, conclusions, and recommendations of this Task Force.

Current clinical and laboratory practice for the investigation of the antiphospholipid syndrome: findings from the 2008 Australasian antiphospholipid antibody survey

The antiphospholipid syndrome (APS) is an autoimmune condition characterised by vascular thromboses and/or pregnancy morbidity, and its diagnosis currently requires laboratory evidence for the presence of antiphospholipid antibodies (aPL). aPL are identified using a large number of laboratory procedures based on one of two distinct test processes, namely 'solid' or 'liquid' phase assays. The former include anticardiolipin antibodies (aCL) and anti-beta-2-glycoprotein-I antibodies (aB2GPI), while the latter are centred on clot-based tests used to identify the lupus anticoagulant (LA). Depending on their clinical presentation, affected individuals might be seen by a variety of clinical specialties including general physicians and general practionners, with a potentially wide variation in the aPL assays requested. The current report summarises findings from the '2008 Australasian antiphospholipid antibody survey', a simple 5-step survey that assessed current clinical and laboratory practice in the investigation of APS. The survey was despatched via various clinical and scientific professional bodies. Responses were received from 130 scientific and clinical personnel, primarily haematology based (94/130; 72%) or immunology based (34/130; 26%). Most respondents (97/130; 75%) ordered or recommended tests for solid phase aPL testing, and most also attempted to grade these tests and their isotypes. Most were familiar with aCL and aB2GPI testing, and tended to request primarily IgG and IgM isotypes of these antibodies. Only a small number of respondents requested/recommended IgA isotype testing of these antibodies or the other solid phase aPL assays (e.g., anti-prothrombin). A similar number of respondents (104/130; 80%) also ordered or recommended tests for LA, and most also attempted to grade these tests and their isotypes. Some discipline-related biases were also evident, in that 32/34 (94%) of immunology-based respondents identified that they ordered or recommended specific solid phase tests for aPL, whereas only 62/94 (66%) of haematology-based respondents did so. In contrast, 83/94 (88%) of haematology-based respondents identified that they ordered or recommended specific LA test procedures, whereas only 18/34 (53%) of immunology-based respondents did so. To our knowledge, this report represents the first ever attempt to survey a wide range of clinical and scientific personnel regarding ordering and recommending practices for aPL testing, and provides a snapshot of current clinical and laboratory practice for the investigation of APS in Australia and New Zealand. Most respondents to our survey still consider the immunoglobulin G (IgG) aCL test to be a useful first-line solid phase aPL test, and the dilute Russell's viper venom time (dRVVT) assay to be the most useful LA test.

Accuracy of the First Fully Automated Method for Anti‐cardiolipin and Anti‐β2 Glycoprotein I Antibody Detection for the Diagnosis of Antiphospholipid Syndrome

In this study we evaluated the diagnostic accuracy for antiphospholipid syndrome (APS) of new fully automated immunoassays for anticardiolipin (aCL) and anti-beta2 glycoprotein I (anti-beta2-GPI) auto-antibody detection (EliA-Phadia), and compared the results with those obtained with Orgentec and Inova ELISA methods. Sixty-two APS patients and 123 controls (20 syphilis, 33 Lyme disease, 30 HCV infection and cryoglobulinemia, 40 healthy subjects) were studied. Using the 99(th) percentile cutoff, the sensitivity and specificity of EliA aCL IgG, aCL IgM, anti-beta2-GPI IgG, and anti-beta2-GPI IgM were 69.4% and 81.9%, 64.5% and 86.7%, 64.5% and 98.8%, and 53.2% and 92.8%, respectively. Using the Sydney criteria cutoff (>40 GPL/MPL units), sensitivity and specificity of EliA aCL IgG and aCL IgM were 45.2% and 98.8%, and 35.5% and 97.5%, respectively. The best diagnostic efficiency was obtained combining the aCL tests (>40 GPL/MPL units) with the anti-beta2-GPI tests (sensitivity 85.7%, specificity 90.4%). The area under the ROC curves for EliA, Orgentec, and Inova methods were 0.870, 0.940, and 0.850 for aCL IgG; 0.820, 0.820, and 0.820 for aCL IgM; 0.910, 0.960, and 0.920 for anti-beta2-GPI IgG; 0.840, 0.840, and 0.820 for anti-beta2-GPI IgM, respectively. Finally, the overall agreement between EliA assays and the other two ELISA methods ranged from moderate (anti-beta2-GPI IgG EliA versus Orgentec: Cohen's k = 0.426) to good (anti-beta2-GPI IgM EliA vs. Inova: k = 0.841). In conclusion, newly developed EliA methods for antiphospholipid antibody detection perform similarly to other ELISA assays and represent a useful tool for APS laboratory diagnosis in daily practice.

The utility of a specific hepatic CYP-3A4 probe (C14-Erythromycin breath test [EBT]) versus the general CYP-P450 probe (antipyrine clearance [ACL] test), for the prediction of docetaxel (D) pharmacokinetics (PK)

2523 Background: BSA-based cytotoxic dosing does not account for the individual variability in drug disposition. In the case of D, CYP 3A4 probes such as the EBT have been assessed to individualise dosing, but inconsistently. This report is the first study comparing the EBT directly with the widely available general P450 probe, the ACL test, for the prediction of D PK when given either q 21 days or weekly. Methods: Patients (pts) with pre-treated advanced malignancy suitable for D therapy, Se bilirubin≤1.0xUNL, AST≤1.5xUNL & ALP≤2.5xUNL, were entered. Prior to D therapy, pts underwent EBT and ACL test. Pts were given IV 14C N-methyl-erythromycin and exhaled breath samples were captured for 14CO2 from 5–120 mins post. The EBT parameters determined: 14CO2 flux at 10 min (CO2f10), & 20 min (CO2f20), (iii) terminal rate constant kCO2 (iv) AUCCO2,(0-∞) & AUCCO2,(0–60). For the ACL test, pts was given oral antipyrine 10mg/kg, blood samples were collected from 0, 4 & 24 hrs post, and serum levels measured: ACL was calculated as per Farrell et al.(Br J Clin Pharmacol 18:559). D was given 75mg/m2 q21 days or 35mg/m2 weekly. Samples taken for D PK in course 1 day 1, parameters included: half life (tD1/2), & clearance (CLD). Correlations were sought between EBT parameters, ACL values and D PK parameters. Results: 20 pts accrued, M:F= 12:8, Median age= 65. Mean BSA = 1.77m2 (1.44–2.07). D q21 days:D weekly= 13:7. EBT parameters (N= 19) (Mean, [CV%]): CO2f10 (%/min) 0.051 (106), CO2f20 0.052 (82), kCO2 (min- 1) 0.007 (22), AUCCO2,(0-∞) 7.9 (85), AUCCO2,(0–60) 2.64 (81). ACL (N=19) (ml/min); 35.8 (37). No significant differences observed for EBT parameters and ACL between the q21 days vs weekly dosing. D PK parameters (N=19): CLD (l/hr) 57.2 (36), tD1/2 (hrs) 12.7 (33). No correlations were observed between the D PK and EMBT parameters for all pts and regardless of the regimen given. For D weekly pts, a significant linear relationship was observed between CLD and ACL (P =0.007, R2= 79.47%). Conclusions: The utility of EBT for the prediction of D PK was not confirmed in this study. The Antipyrine Clearance test may be superior in this regard for D, but regimen dependent and hence warrants further evaluation. No significant financial relationships to disclose.

Clinical relevance of multiple antibody specificity testing in anti-phospholipid syndrome and recurrent pregnancy loss

We wanted to evaluate whether testing for anti-phosholipid antibodies other than anti-cardiolipin (aCL) and anti-beta-2 glycoprotein I (abeta2GPI) immunoglobulin (Ig)G and IgM identifies patients with recurrent pregnancy loss (RPL) who may be positive for anti-phospholipid syndrome (APS). In a cross-sectional study comprising 62 patients with APS, 66 women with RPL, 50 healthy blood donors and 24 women with a history of successful pregnancies, we tested IgM and IgG antibodies to phosphatidic acid, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl inositol and phosphatidyl serine with and without beta-2 glycoprotein I (beta2GPI) from a single manufacturer as well as aCL and abeta2GPI antibodies. Diagnostic accuracies of individual and combined anti-phospholipid (aPL) assays were assessed by computing sensitivities, specificities, positive predictive values and negative predictive values together with their 95% confidence intervals. There was a general trend for increased sensitivities in the presence of beta2GPI co-factor with significant effect for certain specificities. The overall combined sensitivity of the non-recommended aPL assays was not significantly higher than that of the aCL and aB2GPI tests. Multiple aPL specificities in RPL group is not significantly different from controls and therefore of no clinical significance.

A Quarter of a Century in Anticardiolipin Antibody Testing and Attempted Standardization Has Led Us to Here, which Is?

The anticardiolipin (aCL) test has been widely used by physicians since the mid-1980s for diagnosing patients with antiphospholipid syndrome (APS). Establishment of this diagnosis has enabled effective management of patients with recurrent thrombosis and recurrent pregnancy losses. The test was first established in 1983 as a radioimmunoassay and soon thereafter converted into an enzyme-linked immunosorbent assay (ELISA). The other test commonly used in the diagnosis of APS is the lupus anticoagulant (LA) test. The aCL ELISA is sensitive for the diagnosis of APS but lacks specificity. On the other hand, the LA assay, although more specific, is not as sensitive as the aCL ELISA. More specific tests are now available such as the anti-beta2 glycoprotein I (anti-beta2GPI) assay, the antiprothrombin assay, and other ELISAs that use negatively charged phospholipids instead of cardiolipin to coat the plates. In the past 25 years, there have been numerous efforts to standardize aCL, LA, and anti-beta2GPI tests but there are still reports of significant intra- and interlaboratory variation in results for all three assays. This article discusses in detail the clinical value of these tests, technical problems associated with their use, the current laboratory classification criteria for diagnosis of APS, and possible new and better assays that will be available in the near future for diagnosis of APS.

A Consensus Approach to the Formulation of Guidelines for Laboratory Testing and Reporting of Antiphospholipid Antibody Assays

Despite numerous past and ongoing efforts, there remains significant variation in results from assays for the major antiphospholipid antibodies (aPL), namely anticardiolipin (aCL), anti-beta2 glycoprotein I (anti-beta2GPI), and lupus anticoagulant (LA). There is therefore a need to produce comprehensive guidelines on laboratory testing and reporting of aPL assays. However, because of the paucity of good-quality published evidence, there is a heavy reliance on expert opinion, and thus the existing consensus guidelines for aPL testing and reporting are largely eminence based rather than evidence based. This may potentially bias recommendations to reflect the personal preferences of those who have the greatest influence during the guideline formulation process. This article largely details the experience of the Australasian Anticardiolipin Working Party in undertaking a consensus approach to formulation of guidelines on aCL and anti-beta2GPI testing and reporting, including measures taken to minimize these issues. Despite the time-consuming nature of the process, given the paucity of good-quality published evidence, formulation of guidelines by the consensus process remains an important initiative to improve the standardization of aPL testing and reporting.

A protocol for determination of anticardiolipin antibodies by ELISA

The anticardiolipin (aCL) test has been widely used by physicians since the mid-1980s for diagnosing patients with antiphospholipid syndrome (APS). Establishment of this diagnosis has enabled effective management of patients with recurrent thrombosis or recurrent pregnancy losses. The test was first established in 1983 as a radioimmunoassay and soon thereafter converted into ELISA. There have been numerous efforts to standardize the aCL test, but precise reproducible measurement of aCL levels is difficult and the use of semiquantitative measurements (high, medium and low) is recommended as this is probably sufficient for clinical diagnosis. Using validated ELISAs for measuring aCL Abs offers greater reproducibility, would reduce interlaboratory variations and limit discrepancies in results between different laboratories. This article details a procedure that takes approximately 2 h and summarizes the information available on the aCL ELISA test.

Potential Markers of Arterial and/or Venous Thromboses and their Complications in Primary Antiphospholipid Syndrome

Potential Markers of Arterial and/or Venous Thromboses and their Complications in Primary Antiphospholipid SyndromeAntiphospholipid syndrome is characterized by venous or arterial thromboses and/or recurrent abortions accompanied by antiphospholipid antibodies and it can be primary (PAPS) or secondary (SAPS) to another disease. Arterial thromboses are less common than venous and most frequently they manifest as ischemia or infarction. Venous thromboses are usually multiple and bilateral and the most common complication of venous thromboses are pulmonary emboli. Considering that laboratory diagnosis of PAPS is currently based on persistently positive aCL, aβ2gpl and/or LA tests, and that neither one of those tests can discriminate between PAPS patients with arterial or venous thromboses or their complications, the aim of this study was to investigate the diagnostical significance of the determination of apo(a), oxLDL, anti-oxLDL antibodies, antianxA5 antibodies, hsCRP, C3 and C4 complement components and HPT for discrimination between PAPS patients with diverse clinical manifestations. Considering that elevated oxLDL and anti-oxLDL antibodies concentrations were found in PAPS patients, and also in subgroups of PAPS patients with MI or PE, it can be concluded that those parameters represent additional risk factors which together with other factors may lead to thromboses and their complications in PAPS. Regarding the fact that C3 and C4 concentrations were decreased in PAPS patients and that a positive correlation was found between hsCRP and C3 concentrations, this finding could indicate potential roles of these parameters as markers of atherosclerosis, which represents the leading cause of morbidity and mortality. HPT and apo(a) concentrations are not independent risk factors for MI in PAPS because lower levels were found in those patients in comparison to MI survivors without PAPS. No significant correlation of anti-anxA5 antibodies and the presence of arterial or venous thromboses or their complications was found, but increased concentrations of the IgG isotype of those antibodies could be a marker for recurrent abortions in PAPS, although this finding should be further investigated on a larger number of patients with this clinical finding. Determination of hsCRP in PAPS patients could not be an adequate parameter which would provide discrimination between patients with increased risk for development and/or recurrence of venous and/or arterial thromboses, nor for their complications, because no statistically significant difference in concentrations of this parameter was found among PAPS, IM, PE and SLE patients who were included in this study.

Specificity and sensitivity of anti-β2-glycoprotein I as compared with anticardiolipin antibody and lupus anticoagulant in Thai systemic lupus erythematosus patients with clinical features of antiphospholipid syndrome

Antibodies to beta(2)-glycoprotein I (anti-beta(2)-GPI) have been reported to have stronger association with clinical antiphospholipid syndrome (APS) than anticardiolipin antibodies (aCL) and lupus anticoagulant (LAC). We investigated the sensitivity and specificity of ELISA for anti-beta(2)-GPI in Thai systemic lupus erythematosus (SLE) patients with clinical features of APS and compared the results with IgG/IgM aCL and LAC to find the test with the best association. The hospital records of 151 Thai SLE patients whose sera had been sent for either IgG/IgM anticardiolipin antibodies or lupus anticoagulant testing were reviewed. Sera of patients either without complete clinical records or those with APS-related manifestations other than vascular thrombosis and pregnancy morbidity (according to the international consensus statement on preliminary classification criteria for definite APS) were excluded. For the remaining subjects (112 patients), their sera were tested for anti-beta(2)-GPI antibody, IgG and IgM anticardiolipin, and lupus anticoagulant. The sensitivity and specificity of each method were compared by using the chi-square test. Among the 112 (74.2%) SLE patients in the study, 35 (31.3%) presented with preliminary clinical criteria for APS (i.e., vascular thrombosis and pregnancy morbidity) whereas 77 (68.7%) did not. The sensitivity and specificity of anti-beta(2)-GPI determination were 57.1 and 79.2%, respectively, whereas those of IgG aCL were 25.7 and 94.8%, of IgM aCL were 5.7 and 98.7%, and of LAC were 44.8 and 77.3%, respectively. The accuracy of the four tests showed similar association with clinical APS (accuracy of test = 72.3, 73.2, 69.6, and 68.3%, respectively). Concerning the sensitivity, specificity, and difficulty of the methods, the combination of anti-beta(2)-GPI and IgG aCL tests was the best for the diagnosis of APS in Thai SLE patients.

A Review of β2-Glycoprotein-I Antibody Testing Results From a Peer-Driven Multilaboratory Quality Assurance Program

We evaluated the results of lgG beta2-glycoprotein-I (B2GPI) antibody assays in a multilaboratory setting by analyzing data from an external quality assurance program for the 2003 through 2005 cycles for 27 serum samples, including quantitative IgG-B2GPI values and qualitative interpretation and grading (ie, negative or positive; grade of positivity), according to method type and in conjunction with clinical data. We report high interlaboratory variation in numeric IgG-B2GPI results, comparable to that reported for IgG anticardiolipin antibody (aCL) testing, and some method-based variation. For example, interlaboratory coefficients of variation for IgG-B2GPI were more than 50% in 19 samples (70%). For qualitative reporting, there was generally better consensus than previously reported for semiquantitative IgG-aCL testing; although 100% consensus occurred for only 11 samples (41%), more than 90% of laboratories agreed for 19 samples (70%). In some cases, laboratory findings (negative or positive IgG-B2GPI) did not agree with clinical information. Despite the lack of formal standardization for IgG-B2GPI testing compared with IgG-aCL, there seems to be better cross-laboratory consensus. Improvement in standardization of these assays is still required to improve interlaboratory and intermethod concordance of results and interpretation between laboratories and the clinical usefulness of IgG-B2GPI testing.

A Review of ß 2 -Glycoprotein-l Antibody Testing Results From a Peer-Driven Multilaboratory Quality Assurance Program

We evaluated the results of IgG β2-glycoprotein-I (B2GPI) antibody assays in a multilaboratory setting by analyzing data from an external quality assurance program for the 2003 through 2005 cycles for 27 serum samples, including quantitative IgG-B2GPI values and qualitative interpretation and grading (ie, negative or positive; grade of positivity), according to method type and in conjunction with clinical data. We report high interlaboratory variation in numeric IgG-B2GPI results, comparable to that reported for IgG anticardiolipin antibody (aCL) testing, and some method-based variation. For example, interlaboratory coefficients of variation for IgG-B2GPI were more than 50% in 19 samples (70%). For qualitative reporting, there was generally better consensus than previously reported for semiquantitative IgG-aCL testing; although 100% consensus occurred for only 11 samples (41%), more than 90% of laboratories agreed for 19 samples (70%). In some cases, laboratory findings (negative or positive IgG-B2GPI) did not agree with clinical information. Despite the lack of formal standardization for IgG-B2GPI testing compared with IgG-aCL, there seems to be better cross-laboratory consensus. Improvement in standardization of these assays is still required to improve interlaboratory and intermethod concordance of results and interpretation between laboratories and the clinical usefulness of IgG-B2GPI testing.

IgG and IgM anticardiolipin antibodies following treatment with infliximab plus methotrexate in patients with early rheumatoid arthritis

To assess the occurrence of anticardiolipin antibodies (aCL) in patients with early rheumatoid arthritis (RA) receiving treatment with infliximab plus methotrexate (MTX) versus MTX alone. The first 299 patients enrolled in the randomized, Active-Controlled Study of Patients Receiving Infliximab for the Treatment of Rheumatoid Arthritis of Early Onset (ASPIRE) trial who had baseline (week 0) samples available for aCL testing were included in this study. Sera were collected at weeks 0, 30, and 54 from 110 patients taking infliximab 3 mg/kg plus MTX, 98 patients taking infliximab 6 mg/kg plus MTX, and 91 patients taking placebo plus MTX. IgG and IgM aCL were measured using an anticardiolipin assay. IgG and IgM aCL positivity at baseline was similar in all treatment groups. Most patients were negative for IgG aCL at baseline and remained so at the last followup evaluation. One percent (2 of 208) of patients who received infliximab plus MTX and were negative for IgG aCL at baseline were positive for IgG aCL at weeks 30 and 54. A slightly higher proportion of patients who received infliximab plus MTX and were negative for IgM aCL at baseline were positive for IgM aCL at weeks 30 and 54 (4.8% [10 of 208]) as compared with patients who received placebo plus MTX (1.1% [1 of 91]), but the difference was not significant. There was a low incidence of the development of aCL in patients with early RA who received infliximab in combination with MTX, and the difference was not significant compared with patients who received placebo plus MTX.

C‐reactive protein level as a predictor of transient vs. sustained anticardiolipin antibody positivity

The antiphospholipid (APLA) syndrome is defined as the occurrence of venous or arterial thromboembolism or recurrent fetal loss in patients with a positive anticardiolipin (aCL) or lupus anticoagulant test on two occasions, 3 months apart. In this study, we sought to determine whether the level of C-reactive protein (CRP), a non-specific inflammatory marker, at the time of the initial positive aCL test is a predictor of the transient vs. sustained nature of the aCL. Patients with a positive aCL antibody test underwent high sensitivity (hs) CRP testing. A repeat aCL antibody assay was performed on a serum sample obtained 3-4 months later. The positive and negative predictive values of the hs-CRP assay as a predictor of the second aCL result were calculated and correlation between the hs-CRP result and second aCL result was determined. Fifty-four consecutive patients with a positive aCL test were studied. Forty-two had a positive aCL-2. The predictive value of a negative CRP test for a positive aCL-2 was found to be 82.5% (33 of 42 patients). A positive CRP was able to predict a negative aCL-2 in 35.7% of cases (nine of 14 patients). Our results suggest that the hs-CRP test may be useful in the assessment of patients with a positive aCL antibody test. A concurrently performed negative hs-CRP test result may be useful in making diagnostic and therapeutic decisions.

Clinical significance of IgA anticardiolipin and anti-β2-GP1 antibodies in patients with systemic lupus erythematosus and primary antiphospholipid syndrome

The objectives of this study were to estimate the prevalence of IgA anticardiolipin antibodies (aCL) and anti-beta(2)-glycoprotein 1 antibodies (abeta(2)-GP1) in a large number of patients with systemic lupus erythematosus (SLE) and primary antiphospholipid syndrome (PAPS) and to examine possible associations between the clinical manifestations of the APS and the levels of IgA aCL and abeta(2)-GP1. We also assessed the operative characteristics of IgA aCL and abeta(2)-GP1. We retrospectively studied 130 patients with SLE and 35 patients with PAPS. In all patients we measured IgG, IgM, and IgA aCL and abeta(2)-GP1 and recorded any of the clinical manifestations of the APS. IgA aCL were positive in 8.5% of patients with SLE and in 40% of patients with PAPS. Positive IgA abeta(2)-GP1 were found in 17.7% of patients with SLE and in 25.7% of patients with PAPS. IgA aCL were associated with a history of venous thrombosis, thrombocytopenia, and recurrent fetal loss. In contrast, we could not establish significant associations between IgA abeta(2)-GP1 and any of the clinical manifestations of the APS. Measurement of the IgA in addition to IgG and IgM aCL hardly changed the operative characteristics of aCL testing, while measurement of the IgA in addition to IgG and IgM abeta(2)-GP1 increased sensitivity but with a greater loss in specificity. IgA aCL is significantly associated with more than one of the clinical manifestations of the APS in contrast to the IgA abeta(2)-GP1. Routine measurement of the IgA isotype of both aCL and abeta(2)-GP1 does not improve the operative characteristics of aCL and abeta(2)-GP1 and therefore is not recommended at present.

New Facet of Antiphospholipid Antibodies

Since the aCL test was first described, several reports have described the heterogeneity of aPL, which binds to different anionic phospholipids, proteins, or to a phospholipid-protein complex. It has been recently reported that antiphospholipids (aPLs) from the sera of patients with the antiphospholipid syndrome (APS) are able to bind some newly identified antigens, the lyso(bis)phosphatidic acid (LBPA), lipid restricted to the late endosomes, and the sulfatides, acidic glycosphingolipids involved in the hemostatic process. Of interest, aLBPAs are present in the sera of a large number of patients with APS showing similar sensitivity and specificity compared to anti-beta(2) glycoprotein I antibodies (abeta(2)-GPIs) and close association with lupus anticoagulant. Moreover, beta(2)-GPI binds to sulfatides and the majority of the aPL reacting with cardiolipin-beta(2)-GPI complex also react with the sulfatide-beta(2)-GPI complex. Different mechanisms involved in the production of autoantibodies in autoimmune diseases have been proposed and, among them, apoptosis or programmed cell death seems to play a leading role. The relocation of CL and its metabolites during apoptosis may represent an in vivo trigger for the generation of aCL, and the higher reactivity of sera from APS patients to monolysocardiolipin, the immediate degradation product of mitochondrial CL validates this hypothesis. Finally, increasing evidence suggests that oxidative stress could be a pathogenic link between aPL and thrombosis, and antioxidant treatment may have some efficacy in preventing the clinical manifestations of this syndrome.

Development of Consensus Guidelines for Anticardiolipin and Lupus Anticoagulant Testing

Interlaboratory and intermethod variation in commercial and in-house tests used for the measurement of anticardiolipin antibodies (aCL) and lupus anticoagulant (LA) limit the diagnostic value of the results from these tests. This short review summarizes published and unpublished guidelines (some developed using consensus procedures) on aCL and LA testing that are aimed at decreasing assay variation.