Array 360

At ICU admission, peripheral blood samples were also obtained for a routine examination that included complete blood counts, C-reactive protein, procalcitonin, and immunological parameters, which were measured in the PUMCH laboratories, as previously described (29 (link), 30 (link)). In brief, freshly collected EDTA anticoagulated whole blood was incubated and tested with a panel of monoclonal antibodies, then subjected to flow cytometric analysis using a three-color EPICS-XL flow cytometer (Beckman Coulter, Brea, CA, USA) to detect T cells (CD3⁺), CD4⁺ and CD8⁺ T-cell subgroups, B-cells (CD19⁺), and natural killer (NK) cells (CD3−CD16⁺ CD56⁺). Rate nephelometry (Array 360; Beckman Coulter, Brea, CA, USA) was used to measure serum levels of immunoglobulin (Ig)A, IgG, and IgM and of complement factors C3 and C4.

Participants were asked to collect a random morning spot urine sample in a clean, lidded container. All urine samples were assessed centrally in a single reference laboratory at Fifth Hospital, Shanghai, China. Samples were centrifuged at 1500 ×g for 10 min and then stored at −80°C prior to analysis. Further analysis was performed with a Beckman Coulter Array 360 chemistry analyzer. Test results were considered positive for albuminuria (UA⁺) at an albumin-to-creatinine ratio (ACR) >0 mg/g; otherwise the results were accepted as negative (UA⁻; ACR = 0 mg/g). Microalbuminuria, or a moderate increase in UA, was identified as an ACR from 30 to 299 mg/g. Macroalbuminuria was defined as ACR ≥300 mg/g.

C3 and C4 levels were measured by rate nephelometry (Beckman Coulter Array 360, Ramsey, MN, USA). FH and FI levels were measured by radioimmunodiffusion (Binding Site, Birmingham, UK), Screening for FH autoantibodies was undertaken using ELISA as described previously 19. FACS analysis of granulocytes from the patients was performed as described previously 20.

Peripheral blood samples were collected by PUMCH laboratories at enrollment with EDTA anticoagulant for measurement of lymphocyte counts and immunological biomarkers, as described previously (Li D. et al., 2020 (link)). Peripheral blood mononuclear cells were separated, stained with fluorescent monoclonal antibodies, and underwent flow cytometric analysis (3-color EPICSXL flow cytometer; Beckman Coulter, Brea, CA, United States) to detect T cells (CD3⁺), CD4⁺ T cells (CD4⁺CD3⁺ and CD28⁺CD4⁺), CD8⁺ T cells (CD8⁺CD3⁺, CD28⁺CD8⁺), B cells (CD19⁺), and natural killer cells (CD3^–CD16⁺CD56⁺). (Supplementary Figure 1) Rate nephelometry (Array 360; Beckman Coulter) was used to measure serum levels of IgA, G, and M. Inflammatory markers were also recorded, including interleukin (IL)-6, IL-8, IL-10, and tumor necrosis factor (TNF)-α.

Pulmonary function tests were performed according to American Thoracic Society (ATS) guidelines either on the same day as the bronchoscopy or on the day that the serum samples were collected [27 (link)]. Blood gas analysis was performed using a gas analyzer (IL GEM Premier 3000, USA). CRP was performed using ARRAY 360 automatic protein analyzer (BECKMAN, USA). The reference value of serum CRP concentration was 0–10 mg/L. YKL-40 levels were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Uscn Life Science Inc., Wuhan, China). The minimum detection limit of the YKL-40 assay was 13.1 pg/ml.