We have previously reported laparoscopic total biopsy methods for suspected gallbladder cancer (GBC). The present prospective observational study evaluated the safety and feasibility of a novel two-stage algorithm using laparoscopic total biopsy methods. The two-stage algorithm was applied for 40 patients with suspected GBC between July 2018 and September 2022. Laparoscopic whole-layer cholecystectomy (LWLC) was performed for early-stage or suspected malignant lesions without liver invasion and laparoscopic gallbladder bed resection (LGBR) was performed for lesions with an unclear border between the gallbladder and liver. The appropriate strategy could be selected postoperatively depending on the final pathological diagnosis according to examination of permanent sections of gallbladder. If preoperative imaging reveals enlarged lymph nodes (LNs) with possible metastases, LN sampling with intraoperative pathological diagnosis is performed prior to gallbladder removal to determine whether to introduce neoadjuvant chemotherapy. As the first diagnostic procedure, we performed LWLC in 30 cases, LGBR in 8 cases, and LN sampling alone in 2 cases. Median operation time was 165 min and median blood loss was 5.5 ml. No bile leakage caused by intraoperative perforation of the gallbladder was observed. Histologically, GBC was diagnosed in 16 cases (pTis, n = 2
pT1a, n = 2
pT1b, n = 2
pT2, n = 6
pT3, n = 4). Seven of the 10 pT2/3 cases underwent additional open lymphadenectomy. The two-stage algorithm using laparoscopic total biopsy methods for suspected GBC appears to represent a safe, feasible procedure that could play an important role in the optimal treatment strategy.