**2. Materials and methods**

We performed a single-centre, cross-sectional study between March 15th and September 15th 2020. The data from all patients who had tested positive for COVID-19 and had presented with thromboembolic complications were collected in a prospectively maintained database and compared to the data from patients without thromboembolic complications. Inclusion criteria: (a) aged >18 years old; (b) no

*Antiphospholipid Antibodies in Patients with COVID-19 DOI: http://dx.doi.org/10.5772/intechopen.95261*

previous diagnosis of APS (c) positive for COVID-19 (d) patients that required hospitalization. Registry data included the following co-variables: (1) sociodemographic baseline characteristics, such as sex and age; (2) baseline comorbidities including hypertension, dyslipidaemia, hyperuricemia, diabetes mellitus, heart disease, lung disease; (3) need for anticoagulation prior to hospitalization (4) thromboembolic complications during admission (5) mean time of hospitalization (days) (6) laboratorial work (7) aPL positity and isotype determination during hospitalization and confirmed after a 3 month period.

## **2.1 COVID-19 detection**

The diagnosis of SARS-CoV-2 infection was confirmed in all the patients by reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay or serologic testing.

#### **2.2 Analytical parameters**

Screening blood tests included hemogram, D-dimer, C-reactive protein (CRP), procalcitonin, transaminases (serum alanine aminotransferase, serum aspartate aminotransferase), serum lactate dehydrogenase, ferritin, fibrinogen, coagulation times (PTT and aPTT).

#### **2.3 Thrombosis diagnosis**

Computerized tomography was performed to identify COVID-19 thromboembolic related complications. Duplex ultrasound was systematically performed to diagnose proximal and distal lower extremity deep vein thrombosis. Thoracic computer-tomography/angiography was performed if pulmonary embolism was suspected and brain computer-tomography in the case of stroke.

All patients received prophylactic or therapeutic dose low molecular weight heparin (LMWH) (enoxaparin) or unfractionated heparin (UFH), accordingly to thrombotic risk evaluated at admission.

#### **2.4 Detection of aPL**

Determination Serum aCL and aβ2GP1 (IgG, IgM) were determined by the chemiluminescence assay (CIA) (INOVA) and IgG/IgM aPS/PT were determined by ELISA (INOVA).

The detection of LA in human citrated plasma was performed by the HemosIL dRVVT Screen and HemosIL dRVVT confirm assays, as recommended by the International Society on Thrombosis and Hemostasis (ISTH).

aPL positivity was determined at admission and confirmed after 3 months.

#### **2.5 Statistical analysis**

Mann–Whitney U test, χ<sup>2</sup> test, or Fisher's exact test were utilized to compare differences between group A (with thromboembolic complications) and group B (without thromboembolic complications). A two-sided α of less than 0.05 was considered statistically significant. Statistical analyses were performed on SPSS 20.0 package (SPSS Inc.).

Patient dRVVT screen (low phospholipid concentration) and confirm (high phospholipid concentration) results were normalized. Cut-off value was 1.20 for both screen ratio and screen ratio/confirm ratio, demonstrating the

phospholipo-dependence; Anticardiolipin IgG/IgM and/or anti-β2-glycoprotein-I IgG antibodies was defined as elevated if the titer was >20 CU (99th percentile).
