Patients and study design
The development of interstitial lung disease (ILD) in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (CovILD) study is an ongoing prospective multi-centre observational cohort trial aiming to systematically follow patients after COVID-19 (ClinicalTrials.gov number, NCT04416100). A total of 109 patients, aged 18 years or older, who previously suffered from mild to critical COVID-19 were included. All participants gave informed written consent and the study was approved by the local ethics committee at the Innsbruck Medical University (EK Nr: 1103/2020). The inclusion algorithm is depicted in Additional file 1 Fig. S1. Diagnosis of COVID-19 was based on typical clinical symptoms and a positive RT-PCR SARS-CoV-2 result obtained from a nasopharyngeal or oropharyngeal swab. Patients were evaluated 60 days (SD ± 12) after the onset of first COVID-19 symptoms, including clinical examination, medical history assessment, a structured questionnaire to assess typical COVID-19 symptoms, performance evaluation [e.g. six-minute walking test (SMWT)] and the acquisition of blood.
Blood sampling and analysis
Blood samples were taken via routine peripheral vein puncture and analysed by standardized ISO-certified procedures. Additionally, peripheral blood mononuclear cells (PBMCs) were obtained via Ficoll–Paque separation (Pharmacia®, Uppsala, Sweden) from whole blood and EDTA or heparin blood was separated via centrifugation at 300×g to collect serum or plasma, respectively, as previously described in detail . PBMC cell pellets were stored at − 80 °C until further use.
RNA preparation and RT-PCR
We extracted total RNA from PBMC cell pellets using a guanidinium-isothiocyanate-phenol-chloroform-based protocol followed by reverse transcription of mRNA into cDNA, as detailed elsewhere . TaqMan-PCR primers and probes or SYBR-Green primers were designed, and real-time PCR quantification was carried out with Bio-Rad® CFX96 qPCR system using SsoAdvanced™ universal probes supermix (Bio-Rad Laboratories, Hercules, 152 CA). A list of the primers and probes sequences is depicted in Additional file 1 Table S1.
Definition of anemia, iron deficiency and hyperferritinemia
Iron deficiency (ID) was assessed by ferritin, transferrin saturation (TSAT), soluble transferrin receptor and the soluble transferrin receptor/log ferritin index (sTFRF index), as previously described . TSAT < 20% in combination with serum ferritin < 100 µg/L was defined as absolute ID, whereas a TSAT < 20% with serum ferritin > 100 µg/L was considered to reflect functional ID [20, 21].
Anemia was diagnosed according to hemoglobin (Hb) concentrations and gender, whereby a Hb below 120 g/L for women and a Hb below 130 g/L for men were used as cut-offs. The sTFRF index, TSAT and ferritin were used to differentiate between absolute and functional iron deficiency in the setting of anemia [21,22,23]. Accordingly, anemia was categorized as iron deficiency anemia (IDA, sTFRF index > 2, TSAT < 20%, serum ferritin < 30 µg/L), anemia of inflammation (AI, TSAT < 20% and serum ferritin > 100 µg/L or serum ferritin 30-100 µg/L and sTFRF index < 1), a combination of both (IDA + AI, TSAT < 20%, serum ferritin 30-100 µg/L, sTFRF index > 2) or unclassifiable anemia (TSAT normal or reduced, serum ferritin > 30 µg/L, sTFRF index 1–2), as previously described .
Hyperferritinemia was defined by a serum ferritin > 200 µg/L for women and > 300 µg/L for men, as previously reported .
Analysis of lung involvement with computed tomography
60 days after COVID-19 onset, all study participants were evaluated with a low-dose (100 kVp tube potential) computed tomography (CT) scan of the chest. CT was acquired on a 128 slice multidetector CT hardware with a 38.4 × 0.6 mm collimation and spiral pitch factor of 1.1 (SOMATOM Definition Flash, Siemens Healthineers, Erlangen, Germany). CT images were evaluated for the presence of ground-glass opacities (GGO), consolidations, bronchiectasis, and reticulations as defined by the glossary of terms of the Fleischner society . The severity of pathological pulmonary findings was graded for every lobe using the following severity score: 0—none, 1—minimal (subtle GGO, very few findings), 2—low (several GGO, subtle reticulation), 3—moderate (multiple GGO, reticulation, small consolidation), 4—marked (extensive GGO, consolidation, reticulation with distortion), and 5—massive (massive findings, parenchymal destructions). The maximum score was 25 (i.e. maximum score 5 per lobe).
Statistical analyses were performed with statistical analysis software package (IBM SPSS Statistics version 24.0, IBM, USA). Descriptive statistics included tests for homoscedasticity and data distribution (Levene test, Kolmogorov–Smirnov test, Shapiro–Wilk test and density blot/histogram analysis). According to explorative data analysis, we used the following tests: Mann–Whitney U test and Kruskal–Wallis test for group comparisons of continuous data, Fisher’s exact test or Chi-square test for binary and categorical data and Spearman rank test to assess correlations. Multiple testing was adjusted by Sidak formula, as appropriate.