A standardized diagnostic approach is the basis for the treatment of bone and soft tissue tumors. Since bone and soft tissue tumors, especially sarcomas, lack specific symptoms and imaging data provide limited information, biopsy is usually required to clarify the diagnosis. Inappropriate biopsy often brings disastrous consequences to patients, which not only affects limb preservation surgery but also may affect patients’ survival time, so the principles and application of bone and soft tissue tumor biopsy of extremities should be emphasized. 1. Biopsy methods Biopsy methods of bone and soft tissue tumors can be divided into closed biopsy and open biopsy. Closed biopsy is divided into needle aspiration biopsy and core needle biopsy. Open biopsy includes incisional biopsy and excisional biopsy. 1.1 Percutaneous closed biopsy The method of percutaneous closed biopsy was first proposed and applied by Coley in 1931. This method has the following advantages: easy to operate, can be performed in the outpatient clinic or operating room; can be repeated or changed to other biopsy methods; less injury, short healing time, less contamination of the surrounding tissue; rapid, few complications. The main disadvantage is the small size of the pathological tissue obtained. In addition, tissue extrusion, necrotic debris and blood mixing can affect the accurate diagnosis. Needle aspiration biopsy is widely used in the initial diagnosis of primary bone and soft tissue tumors, as well as the diagnosis of local recurrent or metastatic lesions, and can also be used for the diagnosis of lesions in deep sites such as the pelvis under imaging guidance. 1931 Coley et al. first reported the use of needle aspiration biopsy in 35 patients with bone tumors, and the correct diagnosis rate was 70%-90%. The accuracy of needle aspiration biopsy for soft tissue tumors was 64%-96%, and for primary malignant bone tumors was 54%. 95% accuracy of needle aspiration biopsy was reported by Stoker et al. 1.2 Needle biopsy Needle biopsy is also called sleeve biopsy. A core of 3-6 mm in diameter can be obtained by using a trocar needle to extract material deep inside the tumor. This method can also be repeated, and the tissue structure is less damaged. Although the volume of specimen obtained is larger than that of needle aspiration biopsy, it is also more traumatic than needle aspiration biopsy and may cause hematoma and contamination of surrounding tissues. The overall accuracy for diagnosing bone and soft tissue tumors ranged from 68.5% to 95.75%, and Simon briefly described the two puncture methods and the tools used. He concluded that an experienced specialist can achieve 90% accuracy with a 0.7 mm diameter needle and 96% accuracy with a trocar needle with a specimen slot. 1.3 Excisional biopsy Excisional biopsy is the most widely used method for removing tumor tissue under direct vision. The greatest advantage is that sufficient tissue can be obtained for diagnosis and the highest accuracy of histological grading. However, this method has more postoperative complications than closed biopsy. The most common complications are hematoma, tumor spread, incision infection, pathological fracture, especially when the biopsy incision is inappropriately located, which may cause difficulties in whole resection and thus loss of limb preservation. However, even with the risk of hematoma, seroma, and pathological fracture, excisional biopsy is the gold standard in biopsy with a diagnostic accuracy of 98%. 1.4 Excisional biopsy Excisional biopsy is the removal of all the diseased tissue and the sending of pathology at the same time. It is both a diagnostic method and a therapeutic tool. The significance of biopsy at this point is only that the postoperative histological findings confirm the preoperative diagnosis. Excisional biopsy is usually used for soft tissue tumors less than 5 cm and located above the superficial fascia, or small slow-growing bone tumors that can be diagnosed. 2. Principles of biopsy Biopsy of bone and soft tissue tumors should be performed after comprehensive history, physical examination, imaging examination and staging are completed. There are three reasons: (1) Imaging data can determine the characteristics and local size of the tumor and whether it is metastatic or not. (2) Surgical staging of tumor can clarify the anatomical level of the tumor and the invasion area of the tumor. (3) Biopsy followed by imaging may confuse the true tumor boundary and imaging manifestations. Therefore, in principle, biopsy should be performed after the completion of comprehensive imaging examination and staging. Except for some bone and soft tissue tumors that can be clearly diagnosed preoperatively and do not require biopsy. The basic principles of biopsy: (1) Biopsy should be given as much attention as the development of surgical plan before biopsy, and it is best to plan carefully after discussion among doctors from imaging department, pathology department and bone oncology surgery. (2) The principles of aseptic operation, skin preparation, careful hemostasis, non-invasive intradermal suturing or reduction of incisional suture margins should be strictly observed. (3) Ensure that the biopsy does not interfere with the development of future surgical protocols, i.e., the incision or access for open and closed biopsy must be over the surgical incision to be used in the future, so that these tissues and biopsy access that may be contaminated by the tumor can and must be removed intact at the time of the final surgery. (4) The site that best represents the bone and soft tissue tumor is determined prior to biopsy. (5) Biopsy access to the tumor tissue should be as short as possible and must avoid important limb vessels and nerve bundles and not involve too many tissue compartments. (6) Ensure that there are enough representative tissue specimens for the pathologist to make a diagnosis. If the pathologist cannot make a clear diagnosis, detailed clinical and imaging information should be provided in a timely manner. (7) For lesions that cannot be accurately localized on the body surface, there must be imaging equipment to guide them in order to improve the accuracy of puncture and reduce injury and local spread of tumor. (8) If the doctor or hospital is not equipped to diagnose and treat the tumor, the tumor patient should be referred to a doctor or hospital equipped to diagnose and treat the tumor to receive regular diagnosis and treatment before biopsy. Common complications and precautions In 1982, Mankin et al. reported that the misdiagnosis rate of 329 cases of primary malignant tumors of bone and soft tissue biopsies in 16 member centers of the National Musculoskeletal System Tumor Society was 18.2%, and the incidence of complications of skin, bone and soft tissue was 17.3% due to improper sampling sites and biopsy tissues not reflecting the real situation of tumors; In 1996, Mankin et al. conducted a second study to evaluate biopsies in the same way in 597 cases, with a misdiagnosis rate of 13.5%, a complication rate of 15.9%, and unnecessary amputations. The misdiagnosis rate was 13.5%, the complication rate was 15.9%, and the incidence of unnecessary amputation was 3%. Mankin et al. concluded that the risk of bone tumor biopsy is inherent and that newer techniques such as CT and MR are not well suited to reduce it, and that its incidence is primarily related to the bone tumor itself and the surgeon. The risk of bone and soft tissue tumor biopsy by inexperienced surgeons or those who do not have a good understanding of the concept of bone tumor is much higher than that of specialists, as their statistics show that the risk of biopsy in general hospitals is 4 to 12 times higher than that in oncology centers. 25% of patients required expanded resection boundaries and 15% were forced to amputate their limbs. The most common errors in bone tumor biopsy surgery are the use of standard orthopedic surgical approaches and transverse incisions across the mass during surgery, or the use of surgical access to the tumor-associated area across the normal skeletal muscle area or joint. The surgical approach should have a single compartment concept. Common biopsy incision choices for tumor sites are as follows: the medial proximal femur can be accessed through the longus communis muscle and the medial distal femur through the medial femoral muscle; the proximal and lateral distal femur can be accessed through the lateral femoral muscle; the anterior tibia can be accessed through the anterior tibial muscle or directly through the anterior tibial skin; and the proximal humerus tumor should be accessed in the anterior medial third of the deltoid muscle rather than the conventional deltoid-pectoralis muscle gap. The removed tissue specimens are first judged visually, and grayish fish-like tissues are selected to remove blood clots, necrotic tissues and hard bone tissues from them. If the amount of specimens is too small, they can be repeatedly taken several times to ensure that sufficient lesion tissues are obtained. The tissues surrounding malignant tumors are the most vital, representative and diagnostic tissues, and often the center of the tumor is mostly necrotic tissue. If pathological tissues can be obtained from soft tissue masses, it is not necessary to obtain tissues from the bone cortex, because the destruction of the bone cortex often causes pathological fractures and loss of limb preservation opportunities. For fast-growing tumor lesions, it should be avoided to obtain tissue from lesions with high skin tension to reduce the occurrence of wound complications. The incision for incisional biopsy should be as small as possible and sufficient pathological specimens should be obtained. If a transcortical opening to the tumor tissue is necessary, it is advisable to use the smallest possible circular opening or an elliptical opening along the long axis of the backbone to reduce the stress on the bone and reduce the risk of pathological fracture due to biopsy. After the skeletal opening, bone cement filling is needed to avoid the implantation of tumor cells into the surrounding soft tissues. Intraoperative attention should also be paid to the application of instruments to reduce contamination of other tissues, such as avoiding contamination of major blood vessels and nerve bundles during biopsy that could affect later limb preservation surgery. Reduce the application of postoperative drains, as they can be a conduit for malignant tumor dissemination, and if they must be placed, they should be placed near the incision or in the extension of the incision for complete removal at later surgery. Guided biopsy procedures with ancillary imaging facilities will improve the accuracy of the biopsy procedure. The ancillary imaging facilities include X-ray, CT, MRI, ultrasound and FDG-PET. In conclusion, with the deepening of disease understanding, improvement of imaging examination level, improvement of surgical tools and instruments, rational selection of surgical methods for bone and soft tissue tumors according to their different sites and nature and following the principles of bone and soft tissue tumor biopsy of extremities will improve the accuracy of diagnosis and reduce the complications caused by incorrect biopsy methods.