Study design
This study included two parts. The purpose of the first part was to compare the serum SP-A and KL-6 levels between patients diagnosed with IPAF and those diagnosed with non-fibrotic lung disease (non-FLD) and study their diagnostic value. The purpose of the second part was to compare serum SP-A and KL-6 levels before and after treatment and evaluate their prognostic value. The research scheme was approved by the Institutional Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University (ethics approval no. Gyfyy-2016-73).
Diagnostic criteria and treatment
We retrospectively investigated 64 patients with IPAF diagnosed at the First Affiliated Hospital of Guangzhou Medical University between October 2015 and February 2019 according to the diagnostic criteria for IPAF established by the ERS/ATS in 2015. These classification criteria are based on a combination of features from three domains: a clinical domain consisting of extra-thoracic features; a serologic domain with specific autoantibodies; and a morphologic domain with imaging patterns, histopathological findings or multi-compartment involvement. IPAF was confirmed when the patients showed the clinical and/or serological domain criteria specified by the ERS/ATS task force [17].
Sixty-four IPAF patients were initially enrolled: 13 patients (20.3%) met the clinical manifestations and serological manifestations, 16 patients (25%) met the clinical manifestations and morphological manifestations, and 35 patients (54.7%) met the serological manifestations and morphological manifestations. A total of 10 patients (15.6%) met all three criteria.
At present, there is no expert consensus or guidelines on the treatment of IPAF. The treatment approach comes mainly from the approach for connective tissue disease-related interstitial lung disease (i.e., glucocorticoids alone or in combination with azathioprine, cyclophosphamide, pirfenidone and so on).
Among the 64 IPAF patients, 35 were treated with prednisone, 21 were treated with prednisone plus cyclophosphamide, 1 was treated with prednisone plus cyclophosphamide and pirfenidone, 2 were treated with pirfenidone plus cyclophosphamide, 2 were treated with prednisone plus pirfenidone, and 3 were treated with pirfenidone.
Pregnant women, patients with malignant tumours or other autoimmune diseases or co-infections, and patients aged < 18 years were excluded from the study.
Forty-one patients with non-FLD were used as disease controls. Of these 41 subjects, 13 had chronic obstructive pulmonary disease, 10 had lung cancer, 10 had bacterial pneumonia, 2 had eosinophilic pneumonia, 1 had bronchiectasis, 1 had chronic bronchitis, 1 had emphysema, 1 had asthma, 1 had granuloma, and 1 had pulmonary tuberculosis. All diseases met their diagnostic criteria.
The 36 patients with IPAF who received treatment were followed up for > 3 months. The following data were collected from the patients’ medical records: gender, age, body mass index (BMI), smoking history, and lung function.
Lung function measurements
According to the recommendations of the ERS/ATS, lung function tests were performed on a computerized spirometer (MasterScreen, Leibnizstrasse, Hoechberg, Germany). The examination parameters included FVC, forced expiratory volume in 1 s (FEV1), and DLCO.
Blood collection
In the 64 patients with IPAF, the initial symptoms included shortness of breath (41/64, 64.1%), cough (37/64, 57.8%), expectoration (26/64, 40.6%), chest pain and chest tightness (15/64, 23.4%), Dyspnea occurred (5/64, 7.8%) and fever (3/64, 4.7%). Meanwhile, the concomitant symptoms and signs exhibited in some of the patients included inflammatory arthritis and polyarticular morning joint stiffness (8/64, 12.5%), Raynaud phenomenon (2/64, 3.1%), finger swelling (2/64, 3.1%), dry mouth and dry eyes (2/64, 3.1%), muscle soreness (2/64, 3.1%), edema of both lower limbs (1/64, 1.6%), and palpitation (1/64, 1.6%). The symptoms in each patient persisted during the course of the disease.
The fasting morning blood (5 mL) of the patients were collected within 24 hours of the onset of the first respiratory symptoms via coagulation-promoting tubes. The collected samples were stood for about 30 min at room temperature and centrifuged at 3,000 r/min for 10 min to obtain serum. Aliquots of serum were stored at -80℃ to avoid repeated freezing and thawing.
Measurement of serum SP-A and KL-6 levels
Serum SP-A and KL-6 levels were measured on a fully automatic immunoanalyser, HISCL-5000 (Sysmex Corp., Hyogo, Japan), according to the manufacturer’s instructions. The detection range for the SP-A level was 1–1000 ng/mL and that for KL-6 was 10–6000 U/mL. Samples that were above the upper detection limit were excluded from the analysis. SP-A and KL-6 assay kits were obtained from Sysmex Corporation.
Definitions of disease progression, improvement, and stable condition
Disease progression was defined as a decrease in FVC ≥10% and/or DLCO ≥15%. Disease improvement was defined as an increase in FVC by ≥10% and/or DLCO by ≥15%. Stable condition was defined as a change in FVC by <10% and DLCO by <15%.
Statistical analysis
The normality of continuous variables was assessed with the Shapiro-Wilk test, and the data are expressed as the mean ± standard deviation or median plus interquartile range (25–75th percentiles) according to their distribution (normal or non-normal). Dichotomous data are presented as frequencies and percentages. The chi-squared test or Fisher’s exact test was used to analyse the differences in categorical data. Differences in the levels of the various serum markers between subject groups were analysed using the Kruskal-Wallis H test and Wilcoxon’s rank-sum test. Correlation analyses were performed using Spearman’s rank correlation. A receiver operating characteristic (ROC) curve was prepared to analyse the specificity and sensitivity for SP-A and KL-6 for disease activity. All statistical analyses were performed using the SPSS statistical software package for Windows (version 22.0; SPSS Inc., Chicago, IL, USA). P values < 0.05 were considered significant.