Modified bronchoalveolar lavage for macrophage harvest in sheep

Objective: Following lung injury, alveolar macrophages are primed to release inflammatory mediators, in addition to exhibiting decreased phagocytosis and diminished adherence to substrate. At present, no suitable technique exists for harvesting large quantities of macrophages from an entire lobar distribution in sheep. We developed a new technique using commercially available supplies modified for BAL that enables harvesting of large numbers of viable macrophages. Methods: Adult Suffolk ewes (n=4) were anesthetized and endotracheally intubated. A cardiopulmonary bypass retrograde coronary sinus catheter (30.5 cm length, 3 mm internal diameter, with a high volume, low pressure 3 cc balloon 6 mm proximal to the tip) was fused with catheter tubing (31 cm), creating a modified catheter 61.5 cm in length. With a 225 cm guidewire threaded into the lumen, the modified catheter was advanced under fluoroscopic guidance through the endotracheal tube into the lung, until wedged in a lobar bronchus. The guide wire was removed and the balloon at the distal end of the catheter inflated with air, to create a seal. Sterile saline (60 ml) was infused (20 ml/sec) into the lobar bronchus from the proximal end of the modified catheter. The instilled fluid was aspirated (10 ml/sec) using steady gentle suction on the syringe, returning 40 to 45 ml of lavaged fluid which was transferred to a centrifuge tube. The procedure was then repeated in a contralateral lobar bronchus. Cells recovered from the lavage were pelleted by centrifugation (10 minutes at 4°C at 3500 rpm), resuspended in 5 ml of lavage solution and counted with a hemocytometer (Spotlite, American Scientific Products, McGaw Park, IL). Cell viability was determined using trypan blue exclusion. Results: Macrophage recovery ranged from 5 to 10 million and cell viability ranged from 65% to 70%. Each animal was extubated within one hour of the BAL with full recovery. One of three sheep underwent a series of three lavages, with complete recovery. Conclusion: The large lumen and bronchial balloon seal of the modified BAL catheter enables harvesting of 10⁶ macrophages with minimal morbidity, to serially assess pathophysiology of lung injury.