Bf 260

BAL and the analysis including cell counts were performed by professional physicians certified by the Japan Society for Respiratory Endoscopy in Oita University Hospital as described previously (Sakamoto et al., 2004 (link)). After local anesthesia with 4% lidocaine, the patient was premedicated intramuscularly with pethidine hydrochloride (17.5–35 mg). A flexible bronchoscope (BF-260, Olympus, Tokyo) was wedged into the selected bronchopulmonary segment, typically in the middle or lingular lobe for lavage. A 50-mL sterile physiological saline solution at body temperature was instilled through the bronchoscope, and the fluid was immediately retrieved by gentle suction at a reduced pump pressure. Saline instillation was performed two or three times, resulting in 100 or 150 ml in total. The collected BAL fluid was immediately processed, filtered through gauze, and centrifuged at 550 rpm for 5 min. The total cells were counted in a hemocytometer. The slides were stained with May-Grunwald-Giemsa stain, and 900 cells were counted for the cell differentials with microscope objective lens of 40 × or 100 ×.

Flexible bronchoscopy was performed using a fiberoptic bronchoscope (BF‐260, Olympus). EB (at least four specimens) was performed using biopsy forceps in the areas that appeared abnormal, if any, or from secondary carinal areas. Moreover, bronchial brushing was performed before EB. Biopsies were stained for mycobacteria. In addition to the stains, the specimens were also sent for TB RT‐PCR and mycobacterial culture.
The biopsy results were categorized as malignant, TB, and benign (Figure 1). The histopathological diagnosis of TB was based on the findings for epithelioid cells, multinucleated giant cells, or caseous necrosis.7 Benign diagnoses were categorized as specific if a benign neoplasm or specific infection (excluding TB) was diagnosed, and benign diagnoses were classified as nonspecific if the biopsy specimen showed nonspecific benign changes (eg, giant cells, leukocytes, histiocytes, inflammation, or fragments of fibrosis).

PSB samples were collected with a sheath brush (Olympus BC-5 CE, Olympus Imaging, Center Valley, PA, USA) with a distal occlusion composed of polyethylene glycerol through a flexible bronchoscope (Olympus, BF-260). The brush was pushed out of the sheath, cut with ethanol-disinfected scissors, and placed in an Eppendorf tube containing 1.5 ml of saline solution. A part of the PSB samples (0.5 ml) were used for standard bacterial cultures and remaining samples were prepared for total DNA extraction. Simultaneously with PSB sample collection, BAL specimens were obtained by lavaging the airway with approximately 50 ml of 0.9 % NaCl solution through the bronchoscope; and approximately 60 % lavage return volume was collected. Total BALF cells were counted from a 0.05 ml aliquot, 0.5 ml of BALF samples were used for bacterial cultures, and 1 ml of BALF samples were taken for total DNA extraction. Remaining fluid samples were centrifuged (1,000 g for 10 min) at 4 °C, and the supernatant was stored at −80 °C for subsequent cytokine analysis by ELISA. The remaining cell pellets were resuspended with 0.9 % NaCl solution, and a differential cell count was performed using cytospin and Wright-Giemsa staining.

Patient preparation: All patients received local anesthesia using 2% lidocaine spray before undergoing FB. A preoperative assessment was conducted to rule out contraindications. The FB used were Olympus BF-260, BF-P260, and BF-P290. Different bronchoscopes were used according to their availability. Transbronchial biopsy: First, a flexible bronchoscope provided visualization and access to the bronchus. Subsequently, a gentle but precise tissue sampling was performed by opening and closing the biopsy forcesp, capturing a small segment of the lesion. Five to six biopsy specimens were taken from each lesion site. ROSE smear: ROSE slide preparation was conducted by a proficient and professionally trained cytotechnologist based on methods reported in the literature (18 (link)) for each specimen. Each biopsy specimen was spread in a concentric circle with a diameter of 1 centimeter on a sterile cytology slide. The slides were then air-dried and stained using a Diff-Quik stain kit (immersion in A solution for 30 seconds, rinsing with phosphate-buffered saline (PBS), immersion in B solution for 20 seconds, rinsing with PBS), and finally observed under an Olympus BX43 microscope. The remaining tissue after preparation of the ROSE slides was placed in tissue fixative fluid and sent for pathological examination.