Abstract
Inherited germline variants are now recognized as important contributors to hematologic myeloid malignancies, but their reliable detection depends on obtaining uncontaminated germline DNA. In solid tumors, peripheral blood remains free of tumor cells and therefore serves as a standard source for germline testing. In contrast, peripheral blood often contains neoplastic or clonally mutated cells in hematologic malignancies, making it impossible to distinguish somatic from germline variants. This unique challenge necessitates using an alternative, non-hematopoietic tissue source for accurate germline assessment in patients with hematologic myeloid malignancies. Cultured skin fibroblasts derived from punch biopsies have long been considered the gold standard for this purpose. Nevertheless, most existing protocols are optimized for research settings and lack detailed, patient-centric workflows for routine clinical use. Addressing this translational gap, we present a robust, enzyme-free protocol for culturing dermal fibroblasts from skin punch biopsies collected at the bedside during routine bone marrow procedures. The method details practical bedside collection, sterile transport, mechanical dissection without enzymatic digestion, plating strategy, culture expansion, and high-yield DNA isolation with validated purity. By integrating this standardized approach into routine hematopathology workflows, the protocol ensures reliable germline material with minimal patient discomfort and a turnaround time suitable for clinical diagnostics. Key features • This protocol integrates bedside skin punch biopsy with routine bone marrow sampling to minimize patient discomfort and avoid additional invasive procedures. • It uses an optimized enzyme-free mechanical dissection method with fat removal, fine mincing, and scratched well plating to reduce contamination and improve fibroblast yield. • It provides an easy-to-follow workflow for primary fibroblast culture, plating, expansion, and harvest, suitable for routine hematopathology laboratories. • It consistently yields high-quality germline DNA free of hematopoietic contamination, ideal for genetic testing in myeloid malignancies.