Laboratory diagnosis of antiphospholipid syndrome : insights and hindrances

Diagnosis of antiphospholipid syndrome (APS) requires the presence of a clinical criterion (thrombosis and/or pregnancy morbidity), combined with persistently circulating antiphospholipid antibodies (aPL). Currently, laboratory criteria aPL consist of lupus anticoagulant (LAC), anticardiolipin antibodies (aCL) IgG/IgM, and anti-β2 glycoprotein I antibodies (aβ2GPI) IgG/IgM. Diagnosis and risk stratification of APS are complex and efforts to standardize and optimize laboratory tests have been ongoing since the initial description of the syndrome. LAC detection is based on functional coagulation assays, while aCL and aβ2GPI are measured with immunological solid-phase assays. LAC assays are especially prone to interference by anticoagulation therapy, but strategies to circumvent this interference are promising. Alternative techniques such as thrombin generation for LAC detection and to estimate LAC pathogenicity have been suggested, but are not applicable yet in routine setting. For aCL and aβ2GPI, a lot of different assays and detection techniques such as enzyme-linked immunosorbent and chemiluminescent assays are available. Furthermore, a lack of universal calibrators or standards results in high variability between the different solid-phase assays. Other non-criteria aPL such as anti-domain I β2 glycoprotein I and antiphosphatidylserine/prothrombin antibodies have been suggested for risk stratification purposes in APS, while their added value to diagnostic criteria seems limited. In this review, we will describe laboratory assays for diagnostic and risk evaluation in APS, integrating applicable guidelines and classification criteria. Current insights and hindrances are addressed with respect to both laboratory and clinical implications

Evaluation of two assays for monitoring emicizumab and a thrombin generation assay in patients with hemophilia A

Introduction: Emicizumab is a bispecific monoclonal antibody that mimics function of factor (F)VIII. It is effective as prophylactic treatment in hemophilia A with and without presence of inhibitors. Routine monitoring of emicizumab is not required, but can be useful in specific situations. We aimed to compare two assays for monitoring emicizumab and an automated thrombin generation (TG) assay. Methods: We included 33 platelet-poor citrated plasma samples from 15 patients receiving emicizumab. All samples had FVIII levels < 4%. Emicizumab was measured with STA-R evolution® (Stago, France) using a modified one-stage clotting FVIII assay (OSCA) (Stago) and chromogenic FVIII assay (CHROM) (Hyphen Biomed, France), both calibrated with an emicizumab specific calibrator (r² Diagnostics, USA). TG was measured in 15 samples with ST Genesia® (Stago) using STG®-BleedScreen reagent which has a low but unspecified concentration tissue factor as trigger. Results: Emicizumab concentration ranged from 15.6-75.6 µg/mL and 8.98-54.7 µg/mL measured with OSCA and CHROM, respectively. On average, emicizumab concentration was 38.5% (95%CI 34.4-42.6%) lower when measured with CHROM compared to OSCA. Passing-Bablok regression analysis revealed both systematic and proportional differences (intercept: -3.62, 95%CI: -8.65 to -0.50; slope: 0.77, 95%CI: 0.70-0.87). Moderate qualitative agreement between both methods was observed around the therapeutic level of 30 µg/mL (Table 1), expressed as a Cohen’s kappa of 0.51 (95%CI: 0.23-0.79). 12/15 and 9/15 samples with TG analyzed, had emicizumab levels >30 µg/mL for OSCA and CHROM, respectively. All 15 samples generated thrombograms, but TG parameters were mostly outside the in-house calculated reference values. Only one and three samples had peak height (PH) and endogenous thrombin potential (ETP) above the lower reference limit, respectively (Figure 1). No significant correlation was observed between OSCA and TG parameters except for time to peak (r=0.66, p< 0.01). Conclusions: Emicizumab concentration results of the CHROM assay were significantly lower compared to the OSCA results, resulting in different interpretation for 7/33 samples. Thrombin was generated in all samples, measured by ST Genesia, and was lower compared to the reference population although patients had no signs of bleeding. Since no correlation was found with ETP or PH, measures of amount of thrombin generated, TG cannot be used for monitoring emicizumab

Stability of anti-factor Xa activity after sample storage

Introduction: The chromogenic anti-Xa activity assay is used for monitoring low molecular weight heparin (LMWH) and unfractionated heparin (UFH). In daily practice, this test is performed shortly after arrival in the lab, and ideally within four hours of blood collection. For research or method validation purposes, stored samples are of value and citrated platelet-poor plasma may be frozen at -80°C for later analysis. Taking into account the limited scientific literature and the potential benefits of a collection of stored samples, we evaluated this pre-analytical factor. According to literature, it is acceptable to freeze samples with LMWH at -80°C for anti-Xa assays, at least for a limited storage time (up to 24 hours). We investigated whether the anti-Xa effect decreases over a longer time in frozen stored samples (several months) containing LMWH or UFH. Methods: Anti-Xa levels (UFH n=31, LMWH n=30) were compared before and after freezing samples at -80°C for 12–15 months. The STA-Liquid Anti-Xa assay (Diagnostica Stago) was performed on STA Coagulation Analyzers (STA R Evolution® and STA Compact Max®, Diagnostica Stago). Results: Mean anti-Xa activity for UFH and LMWH before storage was 0.38 IU/mL (range: 0.04–0.73) and 0.42 IU/mL (range: 0.03–1.24), respectively. In samples with UFH, the mean anti-Xa activity after storage (one freeze/thaw cycle) was 0.07 IU/mL (range: -0.17–0.19) lower compared to the original measurement (Figure 1). In samples with LMWH, the mean of the anti-Xa activity after storage was 0.01 IU/mL (range: -0.15–0.11) lower compared to the original measurement (Figure 2). For UFH, 6/31 samples were classified differently in regard of the therapeutic range, of which 4 fell outside and 2 within the therapeutic range after freezing. Pearson’s correlation between the first and second anti-Xa activity measurements was 0.93 for samples with UFH and 0.98 for samples with LMWH. Conclusions: We observed a minor mean difference in anti-Xa activity after freezing and thawing heparinized samples. We have shown that a freeze-thaw cycle has little effect on sample stability for LMWH, even for a longer storage period. For UFH, we observed in a limited number of samples an influence on clinical interpretation of anti-Xa levels after sample storage for more than one year

Monitoring of heparin therapy beyond the anti‐Xa activity assay : evaluation of a thrombin generation assay

Introduction: Global coagulation assays may be of added value to the anti-Xa assay for monitoring heparin therapy. Unlike most testing methods, the thrombin generation assay ( TGA) has the ability to assess the overall function of the hemostatic system, which provides information on the anticoagulation status of patients. We compared the TGA, measured with ST Genesia (R) STG-DrugScreen (R) reagent, with the anti-Xa assay for monitoring heparin therapy in inflammatory and non-inflammatory patients. We also determined reference values for STG-DrugScreen (R) thrombin generation (TG) parameters. Methods: Reference values were determined on 120 healthy donors. Furthermore, a spiking experiment with unfractionated heparin (UFH) and low molecular weight heparin (LMWH) was performed, and samples of patients receiving UFH or LMWH were analyzed with ST Genesia (R) and the anti-Xa assay. Results: High discrepancy between TG parameters and anti-Xa activity was observed for low LMWH anti-Xa levels. TG parameters were affected in 36/46 (time to peak) to 42/46 (peak height) patients during UFH therapy with sub-target anti-Xa activity levels. Conclusion: TGA seems insufficiently sensitive for low concentrations of LMWH. There may be an added value of the TGA for monitoring UFH in so-called heparin-resistant patients. Therefore, the TGA has the potential to be introduced as an additional tool for monitoring heparin therapy

Application of the thrombin generation assay in patients with antiphospholipid syndrome : a systematic review of the literature

BackgroundThe antiphospholipid syndrome (APS) is classified by the presence of antiphospholipid antibodies (aPL) and thrombotic and/or adverse obstetric outcomes. The diagnosis and risk assessment of APS is challenging. This systematic review investigated if the thrombin generation (TG) assay could be helpful for APS diagnosis and risk assessment. MethodsA systemic review was performed by searching two databases (MEDLINE and Embase) until March 31, 2022, using a search strategy with two concepts: APS and TG, and related keywords. Two reviewers independently screened the articles based on predefined inclusion and exclusion criteria. Data extraction and quality assessment with the Newcastle-Ottawa Scale (NOS) were performed independently. Synthesis Without Meta-analysis guidelines were followed for data synthesis reporting. ResultsFourteen studies with 677 APS and 1,349 control subjects were included with variable quality according to the NOS. Twelve studies measured TG via the calibrated automated thrombogram (CAT) method using a fluorogenic substrate, whereas two used a chromogenic substrate-based TG assay. One study compared the CAT assay to the fully-automated ST Genesia (R) (Stago, France). Two studies initiated TG using platelet-rich plasma, whereas the rest of the studies used platelet-poor plasma. Resistance to activated protein C (aPC) was examined in ten studies. They reported a significant increase in aPC-resistance in APS patients compared to healthy controls, aPL-carriers, and thrombotic controls. Based on two studies, the prevalence of aPC-resistance was higher in APS patients compared to healthy controls and thrombotic controls with odds ratios of 5.9 and 6.8-12.8, respectively (p < 0.05). In contrast, no significant difference in aPC-resistance was found between APS patients and autoimmune disease controls. Furthermore, 7/14 studies reported TG-parameters including peak height, endogenous thrombin potential, lag time, and time to peak, but these outcomes were highly variable between studies. Furthermore, TG methodology between studies differed greatly, impacting the comparability of the studies. ConclusionaPC-resistance measured with TG was increased in APS patients compared to healthy and thrombotic controls, but the diagnostic and prognostic value is unclear compared to current diagnostic strategies. Studies of other TG-parameters were heterogeneous and more research is needed to identify their potential added value in APS diagnosis. Systematic Review Registrationhttps://www.PROSPERO/, identifier: CRD4202230836

Recherche criminologiques analysée

Le présent ouvrage vous donne un aperçu de la recherche scientifique pertinente en matière de politique qui a été sous-traitée par les Ministres de l'Intérieur et de la Politique scientifique fédérale entre 1995 et 2003. La recherche demandée par la Police fédérale est également reprise. Cette publication reprend la recherche effectuée dans le domaine de la criminologie. Il n'y a pas eu d'aperçu regroupant toute la recherche scientifique sous-traitée dans ce domaine. Sous la direction du Service de la Politique criminelle, un lien de collaboration a été créé en 2000 entre les départements de l'Intérieur, de la Justice et la Politique scientifique fédérale. Ce lien s'est concrétisé par la Plate-forme de concertation pour la Justice et pour la Sécurité. Le présent aperçu est le fruit d'une intense collaboration entre les représentants des services constituant la Plate-forme. Grâce à une concertation constructive et des efforts communs, il a été possible d'atteindre un des objectifs les plus importants de la Plate-forme. La présente publication offre un aperçu transparent de toute la recherche par contrat sous-traité par les autorités au cours des dernières années. La table des matières constitue déjà une petite vue d'ensemble. Une analyse générale de tout le matériel scientifique et une première mise en situation se trouvent au début de l'ouvrage. Vous trouverez ensuite un aperçu schématique reprenant par année les données suivantes: le titre de l'étude, l'université ou institution qui a effectué l'étude, le(s) promoteur(s), la durée, le donneur d'ordre, le coût et la disponibilité du rapport final. Les fiches constituent le cœur de cette publication, rédigées pour chaque étude et se penchant chacune sur la thématique, la problématique posée, la méthodologie de chaque étude. Pour pouvoir offrir cette vision, les rapports d'étude ont été étudiés en profondeur et il y a eu une collaboration intensive entre les services. Dans les parties 2 et 3, ce sont les études ordonnées par le SPF Intérieur, à savoir la Direction Générale Politique de Sécurité et de Prévention et la Police fédérale, qui sont cartographiées. La dernière partie traite des études qui ont été menées dans le domaine de la recherche criminologique ordonnées par la Politique scientifique fédérale. Nous sommes convaincus que le présent Vade-mecum offrira une mine d'informations pour les décideurs politiques, les académiciens et criminologues du domaine criminologique de la prévention, de la justice et de la sécurité

Toward harmonized interpretation of anticardiolipin and anti-β2-glycoprotein I antibody detection for diagnosis of antiphospholipid syndrome using defined level intervals and likelihood ratios : communication from the ISTH SSC Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibodies

Background: Improving harmonization of the clinical interpretation of anticardiolipin (aCL) and anti-beta-2-glycoprotein I (aB2GPI) antibodies immunoglobulin G (IgG)/immunoglobulin M (IgM) in the diagnosis of antiphospholipid syndrome (APS) is desirable. Likelihood ratios (LRs) with corresponding test-result intervals can identify the power of a test to discriminate between a diseased and nondiseased patient and may be useful for the semiquantitative interpretation of aCL/aB2GPI results. Objectives: To determine moderate and high thresholds for aCL and aB2GPI IgG/IgM measured with chemiluminescent immunoassay, enzyme-linked immunosorbent assay, fluorescence enzyme immunoassay, and multiplex flow immunoassay. Methods: aCL and aB2GPI antibodies IgG/IgM were determined with 4 solid-phase systems in a case-control study population including 381 APS patients and 727 controls. Interval-specific LRs (IS-LR) were calculated for ranges determined by prespecified specificity and sensitivity levels. Three methods were used for determining thresholds that separated low, moderate, and high positive antibody levels. Interassay agreement was checked with Cohen's kappa statistics. Results: Assay- and antibody-specific thresholds demonstrated increasing IS-LR, reflecting different clinical significance for low, moderate, and high levels, especially for IgG aCL and aB2GPI and in thrombotic APS. IS-LRs per antibody and unit range were comparable across solid-phase platforms resulting in enhanced harmonization of result interpretation. Agreement between assays for identifying high levels was improved by semiquantitative interpretation compared with that by quantitative reporting. Conclusion: aCL and aB2GPI IgG/IgM moderate and high thresholds were determined for 4 analytical platforms. Thresholds improve harmonized interpretation of aCL/ aB2GPI levels across platforms. The proposed thresholds should be verified in an independent case-control study to check interlaboratory transferability

Added value of antiphosphatidylserine/prothrombin antibodies in the workup of obstetric antiphospholipid syndrome : communication from the ISTH SSC subcommittee on lupus anticoagulant/antiphospholipid antibodies

Background: The added value of antiphosphatidylserine/prothrombin antibodies (aPS/ PT) in the diagnostic workup of antiphospholipid syndrome (APS) is unclear. Currently, diagnosis of thrombotic APS (TAPS) and obstetric APS (OAPS) requires persistent presence of lupus anticoagulant (LAC), anticardiolipin (aCL) immunoglobulin (Ig) G/IgM, or anti-beta 2- glycoprotein I (a beta 2GPI) IgG/IgM antibodies. Objectives: To evaluate the role of aPS/PT IgG and IgM in OAPS. Methods: aPS/PT IgG/IgM, aCL IgG/IgM, a beta 2GPI IgG/IgM, and LAC were determined in 653 patients (OAPS, TAPS, and controls). In-house aPS/PT cut-off values were calculated, titers and prevalence were compared between OAPS, TAPS, and controls and type of pregnancy morbidity. Sensitivity, specificity, likelihood ratios, and odds ratios (OR) with 95% CI were calculated. Results: In OAPS, aPS/PT IgG and IgM showed an OR of 4.32 (95% CI, 2.54-7.36) and 3.37 (95% CI, 1.93-5.89), respectively, but the association was not independent of LAC. Prevalence and titers of aPS/PT IgG and IgM were lower in OAPS than in patients with TAPS. aPS/PT were more prevalent and showed higher titers in patients with late pregnancy loss than in patients with early pregnancy loss with a positivity of 86.4% and 39.3%, respectively. Higher aPS/PT titers did not increase the likelihood of having OAPS. Conclusion: The added value of aPS/PT testing in the current diagnostic workup of OAPS seems limited compared with LAC, aCL, and a beta 2GPI. aPS/PT might be useful in specific subsets of patients with OAPS. However, future multicentric studies are needed to elucidate the risk of less frequent and most severe obstetrical manifestations