Serum Calprotectin as a Potential Predictor of Microvascular Manifestations in Patients with Antiphospholipid Syndrome

This was a single-center cohort study [12]. Consecutive patients referred to Peking Union Medical College Hospital (PUMCH) from June 2012 to September 2022 with persistent aPLs positivity (detected at least 12 weeks apart) and serum samples were included. Serum samples were obtained from patients during follow-up. Follow-up time was defined as the time between the first occurrence of aPLs positivity and the first occurrence of new-onset microvascular manifestations or the last follow-up visit. Follow-up visits ended in November 2022. Blood samples of age- and sex-matched health controls (HCs) were included. Serum calprotectin was measured using an enzyme-linked immunosorbent assay (ELISA) (Elabscience). The cutoff value was defined as mean + 2 standard deviations (SDs) of HCs.

This study was approved by the Medical Ethics Committee of PUMCH (approval number JS-2038) and carried out in accordance with the Declaration of Helsinki and its later amendments. Written informed consent was obtained from all participants. Patients or the public were not involved in the design, conduct, reporting, or dissemination plans of the research.

Blood sampling age, age at the first occurrence of aPLs positivity, gender, smoking history, cardiovascular risk factors, clinical manifestations, and antibody profile of all the patients were collected. Clinical manifestations included thrombosis, pregnancy morbidity, thrombocytopenia, hemolytic anemia, and microvascular manifestations that happened before the last follow-up visit. Microvascular manifestations [1‐3] included APS nephropathy, livedo reticularis, livedoid vasculopathy lesions, valvular lesions, non-stroke CNS manifestations [13], myocarditis, CAPS [14], pulmonary hemorrhage, multiple bone infarction, retinal microvascular manifestations [15], liver infarction [16], cholecystitis, mesenteric vasculitis, and pancreatitis. APS nephropathy was diagnosed by clinical diagnosis or pathology. Livedo reticularis and livedoid vasculopathy lesion were diagnosed by clinical diagnosis. The valvular lesions were diagnosed by transthoracic echocardiography or transesophageal echocardiography. Non-stroke CNS manifestations were diagnosed by magnetic resonance imaging/magnetic resonance angiography (MRI/MRA) or clinical diagnosis. Myocarditis was diagnosed by MRI. CAPS was diagnosed according to the preliminary classification criteria published in 2003 [1]. Pulmonary hemorrhage was diagnosed by computed tomography. Multiple bone infarction was diagnosed by MRI. Retinal microvascular manifestations were diagnosed by ophthalmological examination and fluorescein angiography. Liver infarction, cholecystitis, mesenteric vasculitis, and pancreatitis were diagnosed by pathology. Laboratory data including blood routine examination, immunoglobulin, complement, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) at the time of blood sampling were collected. The treatment regimen including glucocorticoid, anticoagulation, antiplatelet, hydroxychloroquine, and immunosuppression at the time of blood sampling were collected as well.

Lupus anticoagulation (LA), anticardiolipin antibody (aCL), and anti-β2-glycoprotein I antibody (anti-β2GPI) constituted aPLs. LA was measured according to the recommended three-step procedure with two test systems from the Scientific and Standardization Committee for lupus anticoagulant/antiphospholipid antibodies of the International Society on Thrombosis and Hemostasis Subcommittee. Activated partial thromboplastin time-based assay (aPTT) and the dilute Russell viper venom time (dRVVT) were used, and aPTT ratio > 1.20 or dRVVT ratio > 1.20 [17] was defined as positivity. aCL and anti-β2GPI were measured by chemiluminescent immunoassay (CLIA) (iFlash CLIA kits provided by YHLO Biotech Co., Shenzhen, China). Medium or high titer of aCL was defined as a titer > 10 U/mL and medium or high titer of anti-β2GPI was defined as a titer > 20 U/mL according to the manufacturer’s instructions. The detection of aCL and anti-β2GPI showed good sensitivity and specificity in our cohort in the previous study [18].

Continuous variables with normally distributed data were presented as means and standard deviations (SD). Medians and interquartile ranges (IQR) (P₂₅, P₇₅) were used for variables with non-normal distribution. Continuous variables were analyzed with Student’s t test or Mann–Whitney U test. Categorical variables were presented as counts and percentages. Categorical variables were analyzed with Pearson chi-square test or Fisher’s exact test. The prognosis was shown by the cumulative events curve. Further comparisons were performed using a log-rank test. The prediction of calprotectin positivity and microvascular manifestations was analyzed by multivariable logistic regression. Variables included in multivariable logistic regression were selected by least absolute shrinkage and selection operator (LASSO) regression analysis. Forest plots were used to illustrate the risk factors. Pearson correlation analysis was used to detect the correlation between serum calprotectin and other laboratory data. p < 0.05 was considered statistically significant.