Katrien Devreese

Laboratory diagnosis of the antiphospholipid syndrome: a plethora of obstacles to overcome

The antiphospholipid antibody syndrome (APS) is defined by two major elements: the presence in plasma of auto‐antibodies, i.e. antiphospholipid antibodies and the occurrence of clinical features, categorised as vascular thrombosis or pregnancy morbidity. In contrast to recent reviews on the physiopathology of APS, the present review focuses on the laboratory diagnosis of APS. The original clinical and laboratory criteria that defined patients with APS were set in 1998 in the so‐called Sapporo criteria. Although a revision of these criteria was published in 2006, a number of questions on the laboratory diagnosis of APS remain unresolved. The highlight in this review will therefore be on the potential and limitations of the detection of the lupus anticoagulant, as an established laboratory criterion for the diagnosis of APS. The strengths and weaknesses of the current laboratory guidelines are discussed against our current insight in the physiopathology of APS.

Reference Intervals for a Complete Blood Count Determined on different Automated Haematology Analysers: Abx Pentra 120 Retic, Coulter Gen-S, Sysmex SE 9500, Abbott Cell Dyn 4000 and Bayer Advia 120

Abstract We processed 317 samples from healthy adult volunteers for a complete blood count, including leukocyte differentials and reticulocyte parameters, through five new-generation haematology analysers: Abx Pentra 120 Retic, Coulter Gen-S, Sysmex SE 9500, Abbott Cell Dyn 4000 and Bayer Advia 120. From these data nonparametric 2.5–97.5 percentile reference intervals were calculated for all parameters on all analysers. Some differences were found compared with previously reported reference intervals. Reference intervals for platelet parameters and reticulocytes agreed with these usually accepted. For red blood cell parameters, including haemoglobin and haematocrit, and white blood cell count, including absolute white blood cell differentials, our calculated reference intervals were in agreement with less frequently cited earlier reports, but were lower compared to the usually accepted reference intervals.

Challenges in the diagnosis of the antiphospholipid syndrome.

BACKGROUND The antiphospholipid syndrome (APS) is an important cause of acquired thromboembolic complications and pregnancy morbidity. Its diagnosis is based on clinical and laboratory criteria, defined by strict guidelines. The original clinical and laboratory criteria for the identification of APS patients were published in 1999, in the so-called Sapporo criteria. In 2006 these criteria were revised, and recently more precise guidelines for analysis of the lupus anticoagulant have been provided. However, several questions related to the diagnosis of APS remain unanswered. CONTENT In addition to providing a historical perspective, this review covers several challenges in the diagnosis of APS with respect to clinical and laboratory features, while highlighting pathogenic pathways of the syndrome. We discuss ongoing dilemmas in the diagnosis of this complex disease. Although antiphospholipid antibodies are found in association with various clinical manifestations, the older established clinical criteria were not substantively altered in the 2006 update. Several laboratory tests recommended in the latest criteria, including phospholipid-dependent coagulation tests for the detection of the lupus anticoagulant and ELISAs for measuring anticardiolipin and beta2-glycoprotein I antibodies, still show methodological and diagnostic shortcomings. In addition, antiphospholipid antibodies have been described against other antigens, but their clinical role remains uncertain. CONCLUSIONS Despite updated APS criteria, diagnosis of this syndrome remains challenging. Further research on clinically relevant antibodies and standardization of their detection are needed to improve clinical risk assessment in APS.

Thrombotic risk assessment in the antiphospholipid syndrome requires more than the quantification of lupus anticoagulants

Lupus anticoagulants (LACs) are associated with thromboembolic complications (TECs). LACs can be detected by their anticoagulant properties in thrombin generation assays, by the peak height (PH) and lag time (LT). To assess the thrombotic risk in LAC-positive patients, we have expressed the LAC activity quantitatively by PH/LT calibration curves, constructed for mixtures of monoclonal antibodies against beta2-glycoprotein I (beta2GPI) and prothrombin, spiked in normal plasma. PH/LT was determined in LAC patients, with (n = 38) and without (n = 21) TECs and converted into arbitrary LAC units. LAC titers ranged from 0 to 200 AU/mL, with 5 of 59 patients being negative. In the positive LAC titer population (54 of 59), LAC and anti-beta2GPI immunoglobulin G (IgG) titers correlated with TECs, with odds ratios of 3.54 (95% CI, 1.0-1.7) and 10.0 (95% CI, 1.98-50.6), respectively. In patients with single or combined low titers, useful predictions on thrombosis could be made only after additional measurements of soluble P-selectin and factor VII. This layered strategy yielded positive and negative predictive values, sensitivity, and specificity values approximately 90% in this subgroup. Hence, LAC and anti-beta2GPI IgG titers, when combined with selected markers of the hypercoagulable state, allow a relevant thrombotic risk assessment in nearly all patients with LACs.

Influence of dabigatran and rivaroxaban on routine coagulation assays. A nationwide Belgian survey.

The Belgian national External Quality Assessment Scheme performed a nationwide survey using lyophilised plasma samples spiked with dabigatran or rivaroxaban to demonstrate to the Belgian clinical laboratories how these drugs affect their routine coagulation assays prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and antithrombin. Virtually all Belgian laboratories performing routine coagulation testing (189/192) participated in the survey. Both, dabigatran and rivaroxaban significantly prolonged the PT and aPTT in a concentration- and reagent-dependent manner. PT reagents were more influenced by rivaroxaban than by dabigatran and aPTT reagents more influenced by dabigatran than by rivaroxaban. Among PT reagents, Neoplastin R® was the most sensitive to rivaroxaban and Innovin® and Thromborel S® the least sensitive. Converting PT results to INR only increased the variability between reagents. Among aPTT reagents, Actin FSL® was the least sensitive to dabigatran while the other aPTT reagents showed slightly higher sensitivities. The presence of dabigatran led to falsely reduced fibrinogen concentrations when measured with a low thrombin concentration reagent. The presence of dabigatran caused an overestimation of the antithrombin level when measured with a thrombin-based activity assay and the presence of rivaroxaban an overestimation of the antithrombin level when measured with a FXa-based assay. Instrument-related differences were found for all tested parameters. In conclusion, this paper provides detailed information on the effect of dabigatran and rivaroxaban on routine coagulation assays as performed with a large number of reagent/instrument combinations.

Standardization of antiphospholipid antibody assays. Where do we stand

The laboratory criteria (lupus anticoagulants (LA), and/or anti-cardiolipin (aCL) antibodies and/or anti-β2-glycoprotein I antibodies (aβ2GPI)) that define patients with antiphospholipid syndrome (APS) were set in the Sapporo and Sydney criteria published in 1999 and 2006, respectively, and led to a substantial improvement in the recognition of APS. In addition, guidelines for LA detection were published by the Scientific Standardisation Subcommittee (SSC) of the International Society of Thrombosis and Haemostasis (ISTH) in 2009. However, a number of questions on this respect remain unresolved. Recommendations for the aCL and aβ2GPI assays intended to ameliorate the performance of these solid-phase assays. Despite efforts over the years, standardization has not been reached. This review will focus on methodological issues of the three antiphospholipid antibody (aPL) subtypes that are the subject of debate. The use of an international standard for aPL detection might help solve many of the problems caused by a lack of standardization of these assays.

Hemocompatibility of siRNA loaded dextran nanogels

Although the behavior of nanoscopic delivery systems in blood is an important parameter when contemplating their intravenous injection, this aspect is often poorly investigated when advancing from in vitro to in vivo experiments. In this paper, the behavior of siRNA loaded dextran nanogels in human plasma and blood is examined using fluorescence fluctuation spectroscopy, platelet aggregometry, flow cytometry and single particle tracking. Our results show that, in contrast to their negatively charged counterparts, positively charged siRNA loaded dextran nanogels cause platelet aggregation and show increased binding to human blood cells. Although PEGylating the nanogels did not have a significant effect on their interaction with blood cells, single particle tracking revealed that it is necessary to prevent their aggregation in human plasma. We therefore conclude that PEGylated negatively charged dextran nanogels are the most suited for further in vivo studies as they do not aggregate in human plasma and exhibit minimal interactions with blood cells.

Automated indirect immunofluorescence antinuclear antibody analysis is a standardized alternative for visual microscope interpretation

Abstract Background: Screening for antinuclear antibodies (ANA) is a basic tool in the serological work-up of systemic rheumatic disorders. Despite the emergence of alternative screening methods and the difficulties in standardization, indirect immunofluorescence (IIF) remains the recommended method for ANA detection. This study aimed to assess the reliability of automated ANA IIF analysis as a standardized alternative for the conventional manual approach. Methods: ANA testing on HEp-2000 cells was performed on 304 consecutive routine sera, 28 serumbank samples displaying rare staining patterns, 219 samples of well-defined disease cohorts [141 systemic sclerosis (SSc), 13 polymyalgia rheumatica, 22 osteoarthritis, 5 ANCA-associated vasculitis and 38 spondyloarthritis] and 100 healthy donors. All samples were analyzed by automated IIF (Zenit G-sight), by conventional visual IIF microscopy and two ANA screening enzyme immunoassays (EIA). Results: Automated and conventional ANA IIF analysis were comparable for negative/positive interpretation as well as intensity assessment (>90% agreement). In contrast, the accuracy of pattern recognition (26%) was limited. Likelihood ratios (LR) for SSc on results intervals of both Zenit G-sight and EIA increased with increasing level of positivity. Sensitivity within the SSc-associated antibody subsets was higher for Zenit G-sight (97%–100%) than EIA (10%–96%). A significant correlation between the quantitative result obtained by Zenit G-sight and the conventional end-point titer was found. Conclusions: The use of Zenit G-sight for automated ANA IIF analysis offers opportunities to improve standardization. However, a complementary role of the expert technicians remains, especially for pattern recognition and classification of uncertain/negative samples.

Evaluation of a new set of automated chemiluminescense assays for anticardiolipin and anti-beta2-glycoprotein I antibodies in the laboratory diagnosis of the antiphospholipid syndrome

INTRODUCTION The laboratory diagnosis of antiphospholipid syndrome (APS) requires the demonstration of antiphospholipid antibodies (aPL): lupus anticoagulant (LAC) measured through coagulation assays, anticardiolipin IgG or IgM antibodies (aCL) and/or anti-β2glycoprotein I IgG or IgM antibodies (aβ2GPI), usually detected by ELISA. MATERIALS AND METHODS We evaluated the diagnostic value of aCL and aβ2GPI measured by a new automated system using the chemiluminescence principle, the immunoanalyzer Zenit RA (Menarini). RESULTS Results of aCL and aβ2GPI were correlated with the clinical background of the patients and with results of ELISA (n=314). Correlated to the clinical background sensitivity/specificity ranged for aCL IgG between 7.5-45.2% / 54.2-98.8%, for aCL IgM 3.4-5.5% / 89.9-94%, for aβ2GPI IgG 5.5-25.3% / 75.6-100% and aβ2GPI IgM 3.4-4.8% / 89.9-92.3%, depending on the cut-off used. Sensitivity with manufacturers cut-offs was comparable to ELISA, except for aβ2GPI IgG with a significantly lower sensitivity compared to ELISA (5.5% vs 11.6%). In the APS patient population (n=30) sensitivity of aCL IgG and aβ2GPI IgG was higher measured by ELISA compared to Zenit RA (46.7% vs 30.0%, and 46.7% vs 26.7%, respectively). Agreement between Zenit RA results and ELISA results for the four parameters was moderate (Kappa-values ranging 0.509-0.565). Sensitivity was 38.5%, 53.3%, 40% and 69.2% for aCL IgG, aCL IgM, aβ2GPI IgG and aβ2GPI IgM, respectively, applying the highest cut-off value for Zenit RA, raising towards 64.3%, 100%, 57.1%, for aCL IgG, aCL IgM, aβ2GPI IgG, respectively, in a APS patient population. CONCLUSIONS The new technology of chemiluminescense for measuring aPL showed good performance characteristics. Interpretation of results with a cut-off value associated with a good discrimination for disease, resulted in a lower sensitivity for the diagnosis of APS for aβ2GPI IgG measured by Zenit RA assays compared to ELISA; sensitivity for aCL IgG was comparable to ELISA. Specificity for all parameters was high and comparable for aCL and aβ2GPI.

Antiphospholipid antibody testing and standardization

The laboratory criteria that define patients with antiphospholipid syndrome (APS) include lupus anticoagulant (LAC), anticardiolipin antibodies and anti‐β2 glycoprotein I antibodies (aβ2GPI). All assays show methodological shortcomings and the combination of the three tests, each with different sensitivity and specificity, and hence, differences in clinical utility make the laboratory diagnosis of APS challenging. Consensus guidelines and proposals for antiphospholipid antibodies (aPL) testing have been published in the last 20 years and have led to a substantial improvement. Despite efforts so far, standardization is not reached yet, but progress has been made. On‐going efforts to reduce the interlaboratory/interassay variations remain important; even an absolute standardization cannot be feasibly achieved. Taking into account the methodological shortcomings of the means we have available, more detailed guidelines may help in adequate performance of aPL testing. This review will focus on the efforts and achievements in standardization and on the weaknesses and strengths of the current available laboratory methods.