Material
Karnovsky's fixative (0.5% glutaraldehyde + 2.0% paraformaldehyde), hematoxylin, eosin, orange G., Scott's water, xylene, DPX, PBS, poly-L-lysin, epoxy embedding kit, and DAPI were purchased from Sigma chemical company, MO, USA. BSA, ethanol was from Himedia, and Triton X-100 was from Fisher Scientific. Osmium tetroxide was procured from Ted Pella, USA, Uranyl acetate from TAAB, UK, and lead citrate from Ladd. Polyclonal anti-SARS-CoV-2 specific primary antibody (Cat no. ab275759) and Alexa fluor-594 conjugated anti-rabbit secondary antibody (Cat no. ab150080) were purchased from Abcam, Plc, UK.
Methods
Study design and Sample collection
BALF samples were collected from intubated SARS-CoV-2 positive patients in the COVID-19 wards, AIIMS, New Delhi. All samples were collected between 3rd October 2020 and 31st January 2021 (Additional file 1: Supplementary Table 1). The patients were categorized into three groups such as (A) mild infection non-ARDS patients (non-ARDS, n = 8; 2–8 days after first symptoms), (B) moderate infection (n = 8, 9–16 days after first symptoms), and (C) Severe ARDS patients (ARDS, n = 8; 17th day onwards). RT-PCR test was performed to confirm the COVID-19 infection for all the patients recruited in the study.
Sample Processing For The Cellular Enrichment
The BALF sample (15-20ml) was primarily fixed in freshly prepared 20 ml, 2X Karnovsky's solution (final 1% glutaraldehyde + 4.0% formaldehyde) in 0.2M phosphate buffer and stored at 4°C in a COVID-19-designated refrigerator. An on-duty medical physician reviewed and cross-checked all patient's medical records. Patients' demography, symptoms and signs, RT-PCR results, radiographic findings, treatment, supportive care, and survival data were recorded (Additional file 1: Supplementary Table 1).
After the primary fixation, the BALF solution was diluted ten times with 0.1M NaCl solution and strained through a nylon mesh cell strainer with a 100 µm pore. The filtrate was centrifuged at 2500 rpm for 3 min in a swinging bucket, and excess mucus was removed by washing cell pellets (2–3 times for 10 mins) with PBS solution. The cellular content was enriched by centrifugation at 1200g for 5 mins and resuspended again in the primary fixative A (0.5% glutaraldehyde 2.0% paraformaldehyde in 0.1M PB buffer). These samples were processed for PAP staining, IF, SEM, and TEM.
Pap Staining
For PAP staining, cytosmears were prepared using 200 µl of BALF at 800 rpm for 5 min on poly L-lysine coated glass slides and preserved in 90% ethanol. The smears were washed in water for 1 min, and hematoxylin staining was done for 2–4 min with one-minute washing under running tap water, Scott's water, and again tap water, respectively. The smears were gradually dehydrated in 70%, 95%, and 100% ethanol for 2 minutes each, treated with Orange G for 4 min, and progressively dehydrated. The smears were dipped in Eosin stain for 10 minutes and treated with 95% ethanol and acetone for 2 min each. Finally, the stained smears were treated with xylene (3 times, 5 min each) and mounted in DPX.
Immunofluorescence Using Sars-cov-2 Spike Protein-specific Antibody
For the Immunofluorescence study, BALF samples were washed with 0.1 M phosphate buffer (3 times), and smears were prepared with 10 µl of sample on poly-L-lysine coated glass slides and air-dried at RT. Smears were permeabilized by PBST (0.1% Triton X-100 in 1X phosphate buffer saline, PBS) and incubated with a blocking solution (2% BSA in PBS) for 30 minutes. After blocking, it was incubated with the primary antibody (Abcam ab275759, polyclonal antibody against S1 spike protein, dilution 1:500) for four hours in a humid chamber at room temperature. After washing with PBS, fluorophore-conjugated secondary antibody (Alexa fluor-594 conjugated anti-rabbit secondary antibody, Abcam, Cat No.- 150080; dilution 1:500;) was added and incubated for an hour at RT in the darkroom. Smears were washed with phosphate buffer, and DAPI (1 µg/mL) was added and incubated for 5 minutes. Excess DAPI was removed by washing with PBS, and smears were mounted with 90% glycerol. Fluorescence imaging was performed on a laser scanning confocal microscope (Leica SP8 Germany).
Scanning Electron Microscopy
For SEM, enriched and primary fixed cellular components of BALF were dehydrated with ethanol and critical point dried (E-3100, Quorum Tech). Samples were mounted on double-sided tape on the aluminum stubs and were sputter-coated with a gold-based sputter coater (HHV BT-150) for 180 seconds. Electron micrographs were obtained on EVO18 (Zeiss, Germany) SEM operated at 20 kV accelerating voltage, 8–10 mm average working distance with a secondary electron (SE) detector, and magnifications ranging from 5,000X to 30,000X.
Transmission Electron Microscopy
For TEM imaging, enriched cellular constituents of BALF were primarily fixed with 2.5% glutaraldehyde + 2.0% paraformaldehyde in 0.1M phosphate buffer (PB). The cellular pellets were washed with 0.1 M PB (pH 7.4) and post-fixed with 1% osmium tetroxide in 0.1 M PB (pH 7.4) for one hour (secondary fixation) at 4°C. Pellets were washed with distilled water for 2 hours and stained with en bloc staining with 2% uranyl acetate in 50% ethanol. These samples were again washed with distilled water and dehydrated with an ethanol series (50%, 70%, 80%, 90%, and 100%). Dehydrated pellets were infiltrated with toluene/resin and embedded in Araldite CY212 resin. The blocks were polymerized at 65°C for 48 hours. Ultrathin sections (~ 70 nm) were prepared using UC7 ultramicrotome (Leica) and mounted on grids. Sections were stained with 5% uranyl acetate and 5% lead citrate and imaged using Talos F200 Transmission Electron Microscope (Thermo Fisher Scientific).