Protocol for obtaining leukocyte- and thrombocyte-rich fibrin (L-TRF) membrane in domestic quail (Coturnix coturnix japonica)

Autologous hemoconcentrates, such as leukocyte- and platelet-rich fibrin, are widely used in human regenerative medicine; however, standardized protocols for avian species of experimental and clinical relevance remain scarce. To standardize the collection and preparation of leukocyte- and thrombocyte-rich fibrin L-TRF membranes in domestic quail (Coturnix coturnix japonica) and to discuss the clinical and laboratory implications. Fourteen adult quail underwent right jugular venipuncture. Whole blood was collected into additive-free glass tubes and centrifuged at 400 g at approximately 24°C for 5, 10, 15, and 20 min. Membranes were obtained after partial serum absorption on filter paper. Light microscopy using hematoxylin and eosin staining and transmission electron microscopy were performed to characterize the fibrin matrix and cellular components. Mean whole blood volume per bird was 0.89 ± 0.37 mL (~ 0.7% of body weight). Initial viscoelastic membrane formation was observed at ≥ 10 min, with consistent membranes between 10 and 20 min (median 18 min). Macroscopically, final clots showed a layered structure with translucent fibrin, buffy coat, and erythrocyte-rich portions. Microscopy revealed a three-dimensional fibrin network with embedded thrombocytes and leukocytes, predominantly heterophils. Centrifugation of quail whole blood at 400 g for 20 min in dry glass tubes consistently yielded L-TRF membranes, whereas delays > 1 min between collection and centrifugation were associated with membrane failure. Centrifugation of domestic quail whole blood at 400 g for 20 min in plain glass tubes, followed by standardized serum absorption, produced cohesive L-TRF membranes suitable for handling. This low-cost protocol supports future applications in avian regenerative medicine and aids the interpretation of hemoconcentrate-based interventions in comparative clinical pathology.