Introduction

Bone lesions are always challenging, clinically and diagnostically speaking, whether in human patients or pets. Even with the evolution of diagnostic imaging techniques, histopathological confirmation is crucial to determine the etiology of the lesion and biopsy is the first step in this direction. Utilization of guidance modalities improves localization of the lesion and helps directing the instrumental through the lesion, amplifying the chances of acquiring representative samples of the lesion (Samii et al 1999, Saifuddin et al 2000, Vignoli et al 2004, Aoki et al 2005, Wu et al, 2008). Nowadays, in Medicine, computed tomography is the most used guidance modality in bone lesions biopsy but is not the only one (Wu et al, 2008). In certain situations, fluoroscopy and ultrasonography can be used. The use of ultrasound in bone investigations is limited by the material high density which leads to acoustic shadow formation, preventing formation of a clear imaging by sound waves. However, mixed, heterogeneous or mainly lytic lesions which cortical bone integrity is affected allow visualization of the area without acoustic shadow formation as well as the needle to be inserted (Samii et al 1999, Saifuddin et al 2000, Ahrar et al 2004, Lopez et al 2005). The present study proposes the evaluation of ultrasound utilization as guidance modality in the aim to obtain samples from different bone and mineralized lesions in dogs to histopathological diagnosis, pointing its advantages and limitations.

Materials and Methods

Four dogs, all females, from two to eight years, three being from Rottweiler breed and one of non-specific breed (NSB) presented radiographic lesions, one case suggestive of bone infection, two cases suggestive of primary bone neoplasm and one case suggestive of calcinosis. In both dogs with primary bone neoplasm presumptive diagnosis, one Rottweiler and one NSB, lesions were monostotic, localized in femoral distal metaphysis and radial distal metaphysis. The lesion of the dog with presumptive diagnosis of bone infection embraced all metatarsal bones and part of the central tarsal bone. The dog suspected of calcinosis had multiple lesions in one plantar pad foot and adjoining the shoulder joint and the calcaneus without apparent bone involvement. To confirm the presumptive diagnosis, samples of the lesions were collected through percutaneous biopsy with Jamshidi needle 11 gauge using ultrasound guidance. Besides, in the case suspected of bone infection, material was collected to aerobic and anaerobic bacterial culture and systemic fungal culture. All procedures were performed with dogs under general anesthesia and under the owner's authorization, respecting ethical principles in animal experiment and welfare concepts according to the Brazilian Animal Experimental College (COBEA), and with the approval of the Bioethics Committee of the institution where the study was performed. All collected samples were fixed in 10% neutral buffered formalin, processed by routine methods and embedded in paraffin wax. Five micrometers sections were stained with hematoxylin-eosin dye.

Results

Excepting the calcinosis suspicion, all the lesions showed loss of cortical bone integrity (lysis) in different degrees and mild to moderate periosteal reaction. Lesions suspected of calcinosis were apparently localized in subcutaneous tissue and had radiographic imaging compatible with high density granular formations, probably mineralized. The neoplastic lesions had ultrasound imaging of heterogeneous parenchyma and loss of adjacent cortical bone outline, noticing elevation and rupture of several points of the periosteum. Histopathological diagnosis of animals suspected of primary bone neoplasm yielded osteoblastic osteosarcoma, revealing neoplastic cells with ovoid to spindle shaped nucleus surrounded by mineralized trabeculae formations in one case and scarce eosinophilic filiform material compatible with non-mineralized osteoid material in other. The dog suspected of bone infection had only positive anaerobic bacterial culture for Peptostreptococcus sp. Histopathology revealed invading epidermoid carcinoma (also known as squamous cell carcinoma). Lesions suspected of calcinosis were confirmed, revealing some cystic-like structures containing amorphous to granular, darkly basophilic material surrounded by fibrous connective tissue composed of innumerous inflammatory cells. The animal diagnosed with squamous cell carcinoma and one of the osteosarcoma had the biopsy diagnosis compared with surgical specimens from amputation and confirmed.

Discussion

In all the cases, the use of ultrasound as guidance modality allowed visualization of the lesion without significant acoustic shadow formation. It's not only possible to accompanying the needle tract inside the lesion to be biopsied, the waves identified areas of liquid collection compatible with cysts, presence of blood vessels related to the lesion and unstructured areas suggestive of necrosis. The fragments obtained from these areas are known to be one of the main reasons of diagnostic failure of percutaneous biopsy samples (Samii et al 1999, Saifuddin et al 2000, Pramesh et al, 2001, Ahrar et al 2004, Lopez et al 2005). In osteosarcoma cases, ultrasonography was important to identify blood vessels that could increase the risk of local hemorrhage if lesionated and specially localized unstructured areas, posteriorly identified as extensive necrosis areas in macroscopic evaluation of the surgical specimen. This could have contributed to the successful diagnosis obtained in the samples, similarly to what happens in human patients, where ultrasound is routinely used as guidance modality in biopsy of superficial and deep musculoskeletal lesions (Ahrar et al 2004, Aoki et al 2005, Lopez et al 2005). The collection of a sample that is significantly representative of the lesion is the first and fundamental step to the histopathological diagnosis, especially in neoplastic cases, since imaging modalities yield only presumptive diagnosis (Dernell et al, 2001). In squamous cell carcinoma, due to the lytic aspect of the lesion, ultrasonographic view had no obstacles and the hyperechogenicity of the few remaining bone fragments helped directing the needle to these specific areas. This maneuver was important for the slides made from these bone fragments showed invasion of normal bone tissue by the carcinoma while the other slides showed only the carcinoma itself. The bacterial presence in the lesion could be due to the constant licking of the surgical wound by the dog, serving as entrance to this bacteria, component of natural oral cavity environment. Base on histopathological images seen in surgical specimen, it's believed that Peptostreptococcus sp could be present as an opportunistic microorganism, the carcinoma being the main entity responsible for the bone destruction seen. In the calcinosis lesions, the used of ultrasound objective the location of blood vessels which could cause bleeding if ruptured, because in spite of being a superficial subcutaneous lesion, the inflammatory process provoked by the mineral deposition was evident what increases the local vasculature. Besides, it was possible to visualize the granular formations of the lesion whose image was of rounded, hyperecogenic lesions of small dimensions. In this case, no plasmatic alterations were found to justify the lesions, the calcinosis being classified as idiopathic. It's important to salient that the histopathological diagnosis was possible in all samples obtained from Jamshidi needle, granting 100% efficacy. Jamshidi needle percutaneous biopsy samples are known to yield from 90% to 100% of diagnostic efficiency (Dernell et al 2001, Pramesh et al 2001, Vignoli et al 2004, Aoki et al 2005). And it's also a simpler and less expensive procedure compared to the open biopsy and largely used in Medicine (Pramesh et al 2001, Vignoli et al 2004, Aoki et al 2005).

Conclusions

Ultrasonography proved itself a good guidance modality in different kinds of bone and mineralized lesions. Its main advantages are the device portability, making its utilization easy whether in an ambulatory or in a surgery room, and doesn't expose the patient and the team to ionizing radiation. The main limitation of ultrasound is its utilization in intensively proliferating and sclerotic bone lesions which block the action of the waves leading to acoustic shadowing. In these cases, the radiographic guidance is preferred. Percutaneous biopsy, due to its features, can be routinely used in Veterinary Medicine, being an important diagnostic tool to be explored.

References

1.  Samii VF, Nyland TG, Werner LL, Baker TW. 1999. Ultrasound-guided fine-needle aspiration biopsy of bone lesions: a preliminary report. Vet. Radiol. Ultrasound. 40:82-86.

2.  Saifuddin A, Mitchell R, Burnett SJD, Sandison A, Pringle JAS. 2000. Ultrasound-guided needle biopsy of primary bone tumours. J. Bone Joint Surg. 82:50-54.

3.  Vignoli M, Ohlerth S, Rossi F, Pozzi L, Terragni R, Corlazzoli D, Kaser-Hotz B. 2004. CT-guided fine-needle aspiration and tissue-core biopsy of bone lesions in small animals. Vet. Radiol. Ultrasound. 45:125-130.

4.  Aoki J, Koyama Y, Morita H, Takahashi A, Nakajima T, Yagi A, Arai K, Shinozaki T, Watanabe H. 2005. Clinical background and its relation to results of percutaneous needle biopsy of suspected bone metastasis under guidance with CT fluoroscopy. Radiation Med. 23:463-467.

5.  Wu JS, Goldsmith JD, Horwich PJ, Shetty SK Hochman MG. 2008. Bone and soft tissue lesions: what factors affect diagnostic yield of image-guided core-needle biopsy? Radiology. 248:962-970.

6.  Ahrar K, Himmerich JU, Herzog CE, Raymond AK, Wallace MJ, Gupta S, Madoff DC, Morello FA, Murthy R, McRae SE, Hicks ME. 2004. Percutaneous ultrasound-guided biopsy in the definitive diagnosis of osteosarcoma. J. Vasc. Interv. Radiol. 15:1329-1333.

7.  Lopez JI, Del Cura JL, Zabala R, Bilbao FJ. 2005. Usefulness and limitations of ultrasound-guided core biopsy in the diagnosis of musculoskeletal tumours. APMIS 113:353-360.

8.  Dernell WS, Straw RC, Withrow SJ. 2001. Tumors of skeletal system, p. 378-417. In: Withrow SJ & MacEwen EG. Small Animal Clinical Oncology. 3rd edition. W.B. Saunders, Philadelphia.

9.  Pramesh CS, Deshpande MS, Pardiwala DN, Agarwal MG, Puri A. 2001. Core needle biopsy of bone tumours. Eur. J. Surg. Oncol. 27:668-671.