Investigating the Status of Antiphospholipid Antibodies in Patients with Mild COVID-19

¹ Clinical Research Development Unit, Shahid Beheshti Hospital, Hamadan University of Medical Sciences, Hamadan, Iran

² Clinical Research Development Unit, Besat Hospital, Hamadan University of Medical Sciences, Hamadan, Iran

³ Autism Spectrum Disorders Research Center, Hamadan University of Medical Sciences, Hamadan, Iran

⁴ Neurophysiology Research Center, Hamadan University of Medical Sciences, Hamadan, Iran

EXTENDED ABSTRACT

Background

COVID-19 may be associated with coagulation abnormalities and an increased risk of thromboembolic events. Proposed mechanisms include endothelial injury, effects on the immune system, and production of autoantibodies. Antiphospholipid antibodies (aPL), including lupus anticoagulant (LA), anti-cardiolipin antibodies (aCL), and anti-β2-glycoprotein-I (anti-β2GPI) antibodies, have therefore attracted attention in patients with COVID-19. However, previous studies have reported inconsistent findings regarding the prevalence and significance of aPL in COVID-19, and most investigations have focused on severe disease. This case-control study was conducted to determine the status of aPL in patients with definite mild COVID-19 and to evaluate the potential association between mild COVID-19 and these antibodies by comparison with age- and sex-matched healthy individuals.

Methods

This case-control study was conducted at Shahid Beheshti University Hospital in Hamadan, Iran, from October 2020 to November 2021. Sixty patients older than 18 years with definite mild COVID-19 were enrolled. Mild infection was defined by a positive reverse transcription-polymerase chain reaction (RT-PCR) test, peripheral oxygen saturation (SpO2) greater than 94%, temperature below 38°C, and stable vital signs. Patients with a known history of thrombophilia, including systemic lupus erythematosus, rheumatoid arthritis, mixed connective tissue disease, or antiphospholipid syndrome; recently confirmed thrombotic events; active malignancy; pregnancy; or use of therapeutic anticoagulants before COVID-19 were excluded. Sixty healthy individuals matched for age and sex were recruited as controls. Blood samples were obtained from all participants. After centrifugation, serum was stored at −20°C until testing. Serum IgG and IgM antibodies against cardiolipin and β2-glycoprotein-I were measured by enzyme immunoassay using commercial ELISA kits (GA Generic Assays, Dahlwitz, Germany), according to the manufacturer's instructions. The detection threshold for both autoantibody groups was 10 U/mL. Heparinized plasma was used to measure lupus anticoagulant with HEMOCLOT™ LA-S (HYPHEN BioMed); samples were analyzed in duplicate using a CS-2400 analyzer (Sysmex, Japan), and values above 45 seconds were considered positive. Statistical analyses were performed using GraphPad Prism 8. Normally distributed continuous variables were compared with the independent-samples t-test and non-normally distributed variables with the Mann–Whitney test. P<0.05 was considered statistically significant. The study was approved by the Hamadan University of Medical Sciences Ethics Committee (IR.UMSHA.REC.490.1400), written informed consent was obtained, and the study followed the ethical standards of the Declaration of Helsinki and its subsequent amendments.

Results

The study included 60 patients with mild COVID-19 and 60 healthy controls. In both groups, 36 participants (60%) were men and 24 (40%) were women. Mean age was 48.54±15.29 years in the case group and 46.91±16.71 years in the control group, with no significant difference (P=0.57). Serum β2GPI IgG, β2GPI IgM, and aCL IgG levels did not differ significantly between groups. In contrast, serum aCL IgM was significantly higher in patients with mild COVID-19 than in healthy individuals (P=0.004). LA was 35.7 (24.52–46.4) seconds in cases and 36.05 (4.52–40.9) seconds in controls (P=0.88). β2GPI IgM was 1.03 (0.6–63.98) U/mL in cases and 1.00 (0.13–63.0) U/mL in controls (P=0.35). β2GPI IgG was 0.98 (0.56–2.1) U/mL and 0.99 (0.55–12.34) U/mL, respectively (P=0.58). aCL IgM was 1.05 (0.41–4.59) U/mL in cases and 0.83 (0.69–24.46) U/mL in controls (P=0.004). aCL IgG was 2.85 (0.67–11.3) U/mL in cases and 3.26 (0.35–14.67) U/mL in controls (P=0.42). Anti-β2GPI IgG and IgM were positive in two patients; the reported P-values were 0.58 and 0.35, respectively. LA was positive in three of 60 patients and two of 60 controls (P=0.57). aCL was positive in four patients—one with IgG and three with IgM—compared with one control (P=0.004). Thus, the principal statistically significant difference concerned aCL IgM. Overall, most measured aPL parameters did not differ significantly between mild COVID-19 patients and healthy individuals, whereas aCL IgM was more frequent and had a higher measured serum level among patients.

Conclusion

In this study of patients with mild COVID-19, most assessed antiphospholipid antibody parameters were not significantly different from those of age- and sex-matched healthy individuals. However, aCL IgM showed a statistically significant increase in the patient group, and aCL positivity was more frequent among patients than controls. The findings suggest that aPL may occur more frequently in mild COVID-19, although the pattern differs among individual antibody types. Because aPL may have a role in thrombosis, their presence could contribute to coagulation conditions in patients with COVID-19. Further studies are needed to clarify the pathogenic role of aPL and to determine the persistence of these antibodies over time in patients with different degrees of COVID-19 severity.

Keywords: Anti-beta 2 Glycoprotein Antibody; Anti-Cardiolipin Antibody; Anti-Glycoprotein Antibody; Anti-Phospholipid Antibody; COVID-19

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