key: cord-0935759-yfoxndf3
authors: Gendron, Nicolas; Dragon‐Durey, Marie‐Agnès; Chocron, Richard; Darnige, Luc; Jourdi, Georges; Philippe, Aurélien; Chenevier‐Gobeaux, Camille; Hadjadj, Jérôme; Duchemin, Jérôme; Khider, Lina; Yatim, Nader; Goudot, Guillaume; Krzisch, Daphné; Debuc, Benjamin; Mauge, Laetitia; Levasseur, Françoise; Pene, Frédéric; Boussier, Jeremy; Sourdeau, Elise; Brichet, Julie; Ochat, Nadège; Goulvestre, Claire; Peronino, Christophe; Szwebel, Tali‐Anne; Pages, Franck; Gaussem, Pascale; Samama, Charles‐Marc; Cheurfa, Cherifa; Planquette, Benjamin; Sanchez, Olivier; Diehl, Jean‐Luc; Mirault, Tristan; Fontenay, Michaela; Terrier, Benjamin; Smadja, David M.
title: Lupus anticoagulant single positivity at acute phase is not associated with venous thromboembolism or in‐hospital mortality in COVID‐19
date: 2021-04-21
journal: Arthritis Rheumatol
DOI: 10.1002/art.41777
sha: 4fd1855f1a8c499e5763fafdd9363d67d249ced1
doc_id: 935759
cord_uid: yfoxndf3

INTRODUCTION: Antiphospholipid antibodies (APA) clinical relevance in COVID‐19 is controversial. We aimed to investigate the prevalence and prognostic value of conventional and non‐conventional APA in COVID‐19 patients. METHODS: This study was a multi‐centric, prospective observational French cohort of patients hospitalized for COVID‐19 suspicion. RESULTS: 249 patients were hospitalized for suspected COVID‐19, including 154 with confirmed COVID‐19 and 95 not confirmed. We found a significant increase in lupus anticoagulant (LA) positivity among COVID‐19 positive patients (60.9% versus 23.7% in non‐COVID19 patients, p<0.001), while prevalence of conventional (anti‐cardiolipin and anti‐beta‐2‐GP1, IgG and IgM isotypes) and non‐conventional APA (IgA, anti‐phosphatidylserine/prothrombin and anti‐prothrombin IgG and IgM) were low in both groups. COVID‐19 patients with LA positivity had higher levels of fibrinogen (6.0 IQR 5.0–7.0 versus 5.3 g/L IQR 4.3–6.4, p=0.028) and C‐reactive protein (CRP, 115.5 IQR 66.0–204.8 versus 91.8 mg/L IQR 27.0–155.1, p=0.019). Univariate analysis did not show any association between LA positivity and higher risk of venous thromboembolism (VTE, OR 1.02, 95% CI 0.44‐2.43, p=0.95) or in‐hospital mortality (OR 1.80, 95% CI 0.70–5.05, p=0.24). Unadjusted and adjusted (to CRP, age and sex) Kaplan‐Meier survival curves according to LA positivity confirmed the absence of association with VTE or in‐hospital mortality (unadjusted: p=0.64 and p=0.26, respectively; adjusted: hazard ratio = 1.13 95% CI 0.48–2.60 and 1.80 95% CI 0.67–5.01). CONCLUSIONS: COVID‐19 patients have an increased prevalence of LA positivity associated with biological inflammation markers. However, positive LA at admission is not associated with VTE risk and/or in‐hospital mortality.

Coronavirus disease 2019 (COVID-19) is a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and associated with non-specific respiratory syndromes, ranging from mild upper airway symptoms to hypoxemia requiring mechanical ventilation support (1) (2) (3) . An important feature of COVID-19 is the associated-coagulopathy that correlates to disease severity and in-hospital mortality (4, 5) , without any sign of disseminated induced coagulopathy in contrast to previous reports (6) . There is increasing reports of venous thromboembolism (VTE) and arterial thrombosis irrespective of the use of pharmacological thromboprophylaxis (7) (8) (9) (10) (11) (12) (13) (14) . Both macrothrombosis, in particular pulmonary embolism (PE) (15) and microthrombosis into the lungs, have been largely described (16) . Microthrombosis could be consequence of vascular injury and the link between coagulopathy and severity and/or mortality in COVID-19 (17) .

Antiphospholipid syndrome (APS) is an acquired thrombophilia leading to use of long term anticoagulation therapy (18) . Classification of APS requires the presence of one clinical event (thrombosis or pregnancy morbidity) and at least one persistently positive laboratory test for antiphospholipid antibodies (APA), the latter including lupus anticoagulant (LA), anti-cardiolipin (aCL) and anti-beta-2-GP1 (aβ2GP1) IgG and/or IgM (19, 20) . Autoantibodies to phospholipids and phospholipid-binding proteins like anti-prothrombin (aPT), aCL or aβ2GP1 participate to leukocyte and endothelial activation and induce both arterial and VTE. Combination of positive tests in APA profile and particularly triple positivity (LA, aCL, aβ2GP1, same isotype) identifies patients at high risk for thrombosis and allows a more confident diagnosis of APS. Furthermore, very often, triple positive patients are also positive for anti-phosphatidylserine/prothrombin antibodies (aPS/PT), giving additional risk for thromboembolic events to the usual APA profile (tetra-positive patients) (21) . Moreover, APA are not specific to APS but can be detected in healthy individuals and in different clinical setting, including autoimmune conditions, drugs or infectious disease (18) . APA have been largely described during other viral infections (22) and their pathogenicity in these contexts remains controversial. During COVID-19 outbreak, several reports described potential association between APA and thrombotic events (32) . Previous studies exploring LA described between 45% and 88% of positivity in different cohorts in the medical ward and/or intensive care unit (ICU) settings (10, (23) (24) (25) . Only one study suggested in vitro that APA positivity in sera of COVID-19 patients could be prothrombotic but LA testing was not assessed (26) . To the best of our knowledge, there is no large cohort describing complete screening for LA and associated APA.

Moreover, association of APA with VTE or in-hospital mortality in COVID-19 is still a matter of debate.

In the present study, we aimed to investigate the prevalence of conventional and non-conventional APA and explore their relevance according to VTE and mortality outcomes in a large cohort of 249 patients with suspected COVID-19.

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This study was a multicenter, prospective and observational cohort study conducted in a two university hospitals in Paris (France): Hôpital Européen Georges Pompidou and Hôpital Cochin.

From March 14, 2020 to April 20, 2020, patients with suspected SARS-CoV-2 infection were prospectively included. Inclusion criteria were: patients aged over 18 years, presenting with an infectious syndrome and suspected COVID-19, who presented to the emergency department of both hospitals with hospitalization criteria or directly addressed for hospitalization. Suspected COVID-19 had at least one or more symptoms among the following: fever, headache, myalgia, cough, dyspnea, rhinorrhea and digestive symptoms. All suspected COVID-19 patients were tested for SARS-CoV-2 infection by nasopharyngeal swabs and screened for hospitalization criteria based on local guidelines (27) and defined as described in Supplemental Table 1 . Suspected COVID-19 patients fulfilling hospitalization criteria were admitted in dedicated departments (medical ward or ICU), while awaiting laboratory confirmation of SARS-CoV-2 infection. Diagnosis of SARS-CoV-2 infection was confirmed by a positive result of a reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay and/or typical computerized tomography (CT) scan findings of COVID-19 pneumonia.

The study was performed in accordance with the Declaration of Helsinki. All patients provided written informed consent before they were enrolled (SARCODO 2020-A01048-31, NCT04624997).

For all patients included, baseline characteristics (demographic, treatment, cardiovascular risk factors and body mass index, BMI), clinical, biological data and CT-scan evaluations were collected from the medical records using a standardized data collection.

Nasopharyngeal swabs were collected in universal transport medium (Xpert® nasopharyngeal sample collection kit) at hospital admission as previously described (28) . SARS-CoV-2 was detected using Allplex™ 2019-nCoV Assay (Seegene), a multiplex RT-PCR assay that detects in real time three target genes (E gene, RdRP gene and N gene) in a single tube. Data were automatically analyzed using Seegene viewer software. Only qualitative data were considered.

This article is protected by copyright. All rights reserved LA testing was performed by a three-step procedure including screening, mixing, and confirmation. dRVVT using LA1 and LA2 reagents (Siemens, Germany) and aPTT using Automated APTT (Trinity Biotech, Ireland) and a reagent with a weak sensitivity to LA, CK Prest (Diagnostica Stago). The dRVVT assay contains heparin-neutralizer able to quench unfractionated or low molecular weight heparin (up to 1.0 UI/mL) that might lead to false positive detection of LA. In case of LA testing during unfractionated heparin/low molecular weight heparin, anti-FXa activity was quantified and verified to be below the heparin-neutralizer cut-off of 1.0 UI/mL (Supplemental Table 2 ).

aCL and aβ2GP1of IgG, IgM and IgA isotype antibodies were measured in the plasma by BIO-FLASH Chemiluminescent Immuno Assay technology (QUANTA Flash® β 2 GP1 INOVA Diagnostics, Werfen, Les Lilas, France) with a cut-off value (99 th percentile) at 20 AU as previously described (30) . aPS/PT antibodies of IgM and IgG isotype were measured in the serum by ELISAs

This article is protected by copyright. All rights reserved (Quanta Lite, INOVA Diagnostics, Werfen) with a cut-off value (99 th percentile) at 30 AU as previously described (30) . aPT antibodies of IgG and IgM isotype were measured by ELISAs (Orgentec Diagnostika, Mainz, Germany) with a cut-off value (99 th percentile) at 10 AU.

Continuous data were expressed as median (interquartile range, IQR) and categorical data as proportion. Patients were compared according to COVID-19 viral status and to the positivity of LA.

Continuous and categorical variables were compared using respectively Mann-Whitney test and Fisher exact test. In the multivariate analysis, we used logistic regression model to identify risk factor of VTE and in-hospital mortality. The model was adjusted on age, gender and CRP (as binary variable dichotomized according to the median). For the survival analysis, the start of the study was triggered by the diagnosis of SARS-CoV-2 infection and hospitalization. The end of the study was defined either by the death of the patient during the hospitalization or by discharge alive from the hospital. Survival time was calculated as the difference between the date of the diagnosis of SARS-CoV-2 infection and the date of event occurrence (VTE and in-hospital mortality) or the date of hospital discharge. We used Cox proportional Hazard (PH) model adjusted for age, gender and CRP to investigate the relationships between LA positivity and outcomes (VTE or in-hospital mortality).

Kaplan-Meier method was used to represent Cox PH model results according to the positivity of LA.

In the unadjusted survival analysis, survival curves were compared using log rank test.

All analyses were 2-sided and a p-value <0.05 was considered statistically significant. Statistical analysis was performed using R studio software including R version 3.6.3 (R Development Core Team (2019). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria).

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Overall, 249 patients admitted to hospital for suspected COVID-19 were included. Among them, 154 (61.8%) had confirmed COVID-19 whereas 95 (38.2%) did not have COVID-19 and were ultimately found to have other diagnoses (Supplemental Table 3 ). These two groups were not strictly comparable in terms of gender, age, BMI, cardiovascular risk factors, medical history, clinical features and symptoms ( Table 1) . Hence, COVID-19 patients were more often male with increased BMI and with more fever and respiratory symptoms as previously described in COVID-19 (1-3). At admission, when compared to non-COVID-19 patients, COVID-19 patients were more likely to have dyspnea, decreased SpO2, pneumonia on the CT-scan, increased respiratory rate-breath per minute and more subsequently referred to ICU in particular for acute respiratory distress syndrome. In terms of biological features, regarding coagulation disorders, COVID-19 patients had higher median Ddimer levels, longer K-aPTT and lower PT ratio. In COVID-19 patients, fibrin monomers were negative and associated with hyperfibrinogenemia without thrombocytopenia.

LA positivity was assessed at admission of confirmed COVID-19 and non-COVID-19 patients.

When compared to non-COVID-19 patients (Figure 1A) , we found higher prevalence of LA positivity among confirmed COVID-19 patients (60.9% versus 23.7%, p<0.001). Interestingly, among all COVID-19 patients tested for LA, 9 (7.8 %) received hydroxychloroquine at admission and among them, 6 (75.0%) had LA positivity and 3 (25.0%) LA negativity.

The prevalence of solid phase immunoassays for conventional and non-conventional makers of APS in COVID-19 patients and non-COVID-19 patients is described in Table 2 . Regarding aCL positivity, IgG, IgM and IgA were low in both groups, respectively, 3.2%, 7.4% and 2.1% in non-COVID-19 patients versus 5.8%, 1.3% and 1.9% in COVID-19 patients. aCL IgM were significantly more frequent in non-COVID-19 patients (p=0.008). aβ2GP1 positivity, IgG, IgM and IgA were low in both groups, respectively found in 0.0%, 4.2% and 2.1% in non-COVID-19 patients versus 3.2%, 1.9% and 1.3% in COVID-19 patients. aβ2GP1 IgA were significantly more frequent in non-COVID-19 patients (p<0.001). aPS/PT positivity, IgG and IgM were 0.0% and 10.5% in non-COVID-19 patients, respectively, compared to 0.0% and 4.5% in COVID-19 patients, without any significant difference between groups. Finally, IgG and IgM aPT positivity was 7.4.0% and 5.3% in

This article is protected by copyright. All rights reserved non-COVID-19 patients, respectively, compared to 7.1% and 6.5% in COVID-19 patients, aPT IgM were significantly more frequent in COVID-19 patients (p=0.003). Among COVID-19 patients with LA positivity (n=70), 62 (88.6%) were isolated and 8 (11.4%) were associated with ≥1 other APA (aCL and/or aβ2GP1 and/or aPS/PT, IgG or IgM, Figure 1B) .

In COVID-19 patients, those with LA positivity or not were comparable in terms of gender, age, BMI, cardiovascular risk factors, medical history and time from illness onset to hospitalization ( Figure 2A ) even after adjustment to CRP, age and sex ( Figure   2B ), or the risk of in-hospital mortality (p=0.26, Figure 2C ) even after adjustment to CRP, age and sex ( Figure 2D ).

This article is protected by copyright. All rights reserved Discussion COVID-19-associated coagulopathy is associated with microthrombosis, VTE and arterial thrombotic complications (14, 15, 31) . To the best of our knowledge, the present study is the first one testing all APA in a large cohort of suspected COVID-19 patients, including both confirmed and not confirmed COVID-19. This study explored the relevance of conventional and non-conventional APS markers at COVID-19 admission, to assess whether they might play a role for the prognosis of the disease. As However, a major flaw of this study is the absence of APA-specificity of the COVID-19 patients purified IgG. In this latter study LA testing was not assessed.

APA can be transitory positive in patients with infectious conditions (22) and these antibodies are rarely associated with thrombotic events, explain why this association cannot be reliable in critically ill patients. Whether COVID-19 APA are similar to the ones found in other infectious diseases such as HCV, HBV and HIV remains to be determined (18, 22) .

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In our study, we demonstrated that LA positivity in COVID-19 patients was not associated with more VTE, in particular PE, or with a poorer prognosis. Our results are in accordance with previous reports on smaller cohorts suggesting the lack of association between APA and COVID-19 severity and/or VTE (24, 25, 36) . The high prevalence of stroke (13) or VTE in severe COVID-19 (15, 30) , in particular PE, is unusual and has rarely been reported in other viral infections as influenza virus (8) .

In the study by Devreese et al (37) , 10 COVID-19 patients were tested again one month after the first testing and all but one patient initially positive for LA became negative. This latter report reinforces the hypothesis that LA may be transient and/or artefactual due to acute phase of infection and increased CRP and fibrinogen levels. Furthermore, Pengo et al (38) showed that in suspected APS patients, the initial single APA positive phenotype was confirmed in only 40% of subjects. LA are heterogeneous antibodies detected under various clinical circumstances where cellular damage, due to infectious, autoimmune or inflammatory stimuli, leads to plasma membrane remodeling including release of membrane microparticles and exposure of anionic phospholipids. LA activity may be induced by aβ2GP1 and/or aPT antibodies that provoke a dimerization of β2GP1 and/or prothrombin enhancing their affinity for negatively charge phospholipid (39) . Strikingly, such high prevalence of LA/APA in of COVID-19 patients have rarely been observed with other pathologies, which probably reveals significant or massive cellular destruction specific to COVID-19.

Medium/low APA titers were consistently found in COVID-19 patients. We acknowledge that in the present study, APA testing were performed during the acute phase what is discouraged in the guidelines because of potential interference and guidelines recommend retesting after 3 months to avoid overdiagnosis by classification of transient positivity of APA (19, 20, 33) . Of note, heparin therapy was not an issue in our study for LA testing because our reagents contain heparin neutralizers and anti-FXa activities in patients anticoagulated with heparin were below the cut-off of the neutralizer.

Limitation of our study is the small sample size of both groups and heterogeneity of our non-COVID-19 control group.

In summary, our study demonstrates that COVID-19, similarly to other acute infectious inflammatory diseases, has high prevalence of LA positivity, but the latter is not associated with more VTE and/or in-hospital mortality. LA and APA testing is not recommended and must be discouraged during the acute phase of COVID-19 as for other viral infections. A biological confirmation should anyway be necessary after recovery. In COVID-19, LA did not predict VTE (A) even after adjustment on CRP, age and sex (B). In COVID-19, LA did not predicted in-hospital mortality (C) even after adjustment on CRP, age and sex (D).

LA: lupus anticoagulant, VTE: venous thromboembolism, HR: hazard ratio. 

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This article is protected by copyright. All rights reserved 

Clinical Characteristics of Coronavirus Disease 2019 in China

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia

Curative anticoagulation prevents endothelial lesion in COVID-19 patients

Acute Pulmonary Embolism in COVID-19 Patients on CT Angiography and Relationship to D-Dimer Levels

Pulmonary Embolism in COVID-19 Patients: Awareness of an Increased Prevalence

Confirmation of the high cumulative incidence of thrombotic complications in critically ill ICU patients with COVID-19: An updated analysis

High risk of thrombosis in patients with severe SARS-CoV-2 infection: a multicenter prospective cohort Accepted Article This article is protected by copyright. All rights reserved study

Incidence of venous thromboembolism in hospitalized patients with COVID-19

High incidence of venous thromboembolic events in anticoagulated severe COVID-19 patients

Risk of Ischemic Stroke in Patients With Coronavirus Disease 2019 (COVID-19) vs Patients With Influenza

Venous and arterial thromboembolic complications in COVID-19 patients admitted to an academic hospital in

Prevalence and characteristics of pulmonary embolism in 1042 COVID-19 patients with respiratory symptoms: A nested case-control study

Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis in Covid-19

Respiratory mechanics and gas exchanges in the early course of COVID-19 ARDS: a hypothesis-generating study

Antiphospholipid syndrome

International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS)

Laboratory criteria for antiphospholipid syndrome: communication from the SSC of the ISTH

Additional laboratory tests to improve on the diagnosis of antiphospholipid syndrome

Risk of developing antiphospholipid antibodies following viral infection: a systematic review and meta-analysis

Lupus anticoagulant is frequent in patients with Covid-19

Are antiphospholipid antibodies associated with thrombotic complications in critically ill COVID-19 patients?

High Prevalence of Acquired Thrombophilia Without Prognosis Value in Patients With Coronavirus Disease

Prothrombotic autoantibodies in serum from patients hospitalized with COVID-19

Angiopoietin-2 as a marker of endothelial activation is a good predictor factor for intensive care unit admission of COVID-19 patients

Nasal swab sampling for SARS-CoV-2: A convenient alternative in time of nasopharyngeal swab shortage

Update of the guidelines for lupus anticoagulant detection

Prevalence and Significance of Non-conventional

Risk of venous thromboembolism in patients with COVID-19: A systematic review and meta-analysis

The tissue thromboplastin inhibition test in the detection of lupus anticoagulants: importance of a correction factor eliminating the influence of fibrinogen level

Guidance from the Scientific and Standardization Committee for lupus anticoagulant/antiphospholipid antibodies of the International Society on Thrombosis and Haemostasis

Coagulopathy and Antiphospholipid Antibodies in Patients with Covid-19

Anticardiolipin IgG autoantibody level is an independent risk factor for COVID-19 severity

Anti-Phospholipid Antibodies in COVID-19 Are Different From Those Detectable in the Anti-Phospholipid Syndrome

Antiphospholipid antibodies in patients with COVID-19: A relevant observation?

Confirmation of initial antiphospholipid antibody positivity depends on the antiphospholipid antibody profile

Complexes of anti-prothrombin antibodies and prothrombin cause lupus anticoagulant activity by competing with the binding of clotting factors for catalytic phospholipid surfaces

InteractiVenn: a web-based tool for the analysis of sets through Venn diagrams

Interleukin-6 -pg/mL, median

Outcomes ICU patients -n (%)

Symptomatic PE -n (%)

This article is protected by copyright. All rights reserved Deceased -n (%)