Shaaban, HebatAllah M.a; Aly, Ahmed M.b; Abou-Sinna, Imana
As reported in the literature, although most bone tumors can be diagnosed on the basis of plain radiographs (Springfield and Rosenberg, 1996), biopsy is an important aspect of the preliminary investigation; open biopsy is the considered procedure of choice for diagnostic tissue sampling but it requires hospitalization and sometimes contaminates surrounding tissues, with an associated risk of infection, hematoma formation, and pathological fracture (Gogna et al., 2008).
Fine-needle aspiration cytology (FNAC) is gaining increasing popularity in the diagnosis of musculoskeletal lesions, and in many patients, a definitive diagnosis can be rendered from aspiration smears alone (Nnodu et al., 2006). Precise classification of a tumor is difficult on the basis of FNAC alone and histopathological confirmation is frequently required. However, FNAC of bone lesions has its own advantages of being simple, safe, and inexpensive. It is a quick outpatient procedure, does not require hospitalization, and allows preliminary diagnosis within 15–20 min of aspiration (Wahane et al., 2007). It can also be repeated at different sites in case of inadequate material being aspirated. In addition, FNA can be performed safely in difficult sites such as the vertebrae or pelvis, and adjunctive methods such as electron microscopy, immunocytochemistry, cytogenetics, and DNA-ploidy studies can be used in an attempt to arrive at a definitive diagnosis (Jorda et al., 2000). This study was carried out to assess the accuracy of FNAC in the diagnosis of bone lesions and to highlight some of the challenges. Special attention was paid to examine the utility of FNAC in the preoperative diagnosis of bone lesions and its impact on therapeutic decisions.
Patients and methods
This study was carried out by the Cytology Unit in conjunction with the Radiodiagnosis Department, National Cancer Institute, Cairo University, Cairo, Egypt, between May 2009 and December 2010.
The study included 47 male and 38 female patients whose ages ranged from 4 to 68 years. Most of the patients (38.5%) were in their second decade of life. Cases were selected on the basis of their clinical features (bony swelling, pain, pathologic fracture) or radiologic recognition as bone tumors. FNAC was carried out by the study cytopathologists using palpation (43 cases) or the study radiologist using image guidance (42 cases). The exact site of aspiration and the depth of swelling were first determined by carefully studying anteroposterior and lateral views of radiographs. The aspirations were carried out using either 23- or 25-G disposable needles attached to a disposable 10 ml plastic syringe and received onsite immediate evaluation by a cytopathologist for adequacy assessment. The aspirates obtained were spread on frosted-ended glass slides and immediately fixed in 95% ethanol alcohol for modified Papanicolaou staining. If there was sufficient material, a cell block was prepared after centrifugation. Wherever necessary, an entire battery of antibodies was available for the comparative immunohistochemical (IHC) study, especially utilizing cell block preparations. IHC was ordered in an effort to classify the cells present (epithelial, hematolymphoid, mesenchymal, etc.) and to subtype the lesion when possible. At the time of final interpretation, the modified Papanicolaou-stained smears, in addition to the hematoxylin and eosin-stained sections of the available cell block, were evaluated. The provisional diagnosis was interpreted meticulously in conjunction with the results of the IHC studies available to render a final diagnosis. The cytological diagnoses were classified into four categories: (a) positive for malignant cells including determination of the type of malignancy (carcinoma, sarcoma, lymphoma, myeloma, etc.) and whether it was primary or metastatic; (b) suspicious for malignancy or neoplasia; (c) benign, borderline, or inflammatory lesions; and (d) inadequate or less than optimal for evaluation. A diagnosis of ‘suspicious for malignancy’ was rendered when abnormal cells were observed, but the diagnostic material was limited for a definitive diagnosis of malignancy. A diagnosis of ‘inadequate’ was rendered when smears were acellular or consisted of blood despite repeated aspiration. The findings of FNAC were compared with the subsequent available histologic diagnoses and with the available ancillary techniques. Wherever possible, an attempt was made to render an exact definite cytological diagnosis.
The overall clinicopathological characteristics of the 85 cases are summarized in Table 1. Of 85 cases (47 men, 38 women, mean age 36, age range 4–68 years) seen between May 2009 and December 2010, material sufficient for diagnosis was obtained in 81 (95.3%) patients. Radiologically, 13 (87%) of the 15 predominantly lytic lesions, all of the 43 (100%) mixed lytic and sclerotic lesions and 25 (92.6%) of the 27 predominantly sclerotic lesions, yielded adequate material for cytological interpretation. The most frequent adequately aspirated site was the femur. The vertebrae were the most frequent bones that yielded inadequate samples. Detailed cytologic diagnoses were given only after complete study and correlation of the clinical, radiological, and cytological features.
Correlation of the cytologic diagnoses with the histopathologic, immunocytochemical, and serum electrophoresis findings was available for 66 of the 85 cases studied cytologically (77.6%), and included 44 cases that were cytologically labeled ‘malignant’, nine cases that were labeled ‘suspicious’, four cases that were labeled ‘inadequate,’ and nine cases that were cytologically labeled ‘benign and borderline or inflammatory’. Correlation with histopathologic examination was available in 44 cases (66.6%), with immunohistochemistry in 19 cases (28.8%), which was applied either on the available cell block material or directly on cytological smears and by serum electrophoresis [three cases of plasma cell myeloma (4.6%)] (Table 2). Cytologic diagnosis was confirmed in the 66 patients with available correlation, yielding a diagnostic accuracy of (91%) (Table 3). The diagnostic errors in this study occurred in two cases: a case of malignant fibrous histiocytoma (MFH) that was reported as ‘osteosarcoma’ and a case of metastatic lobular carcinoma that was reported as ‘chondrosarcoma’. All nine cases (10.6%) cytologically diagnosed as ‘suspicious for malignancy’ proved to be malignant lesions; two were osteosarcomas, two were angiosarcomas, one was fibrosarcoma, and four were metastatic neoplasms (one case from each, prostatic adenocarcinoma, colonic adenocarcinoma, follicular thyroid carcinoma, and small-cell carcinoma of lung origin).
Details of discordant cases
In one patient, a cytologic diagnosis of ‘osteosarcoma’ was made but the histopathologic examination proved to be MFH. The patient was a 19-year-old male with a mass in the lower femur. Cytology correctly identified this as ‘malignant’ but the type was not determined. The histopathologic examination has shown that the foci of neoplastic osteoid seen in the fine-needle aspiration smears were in fact irregular coarse collagen fibers. In the second patient, a pelvic metastatic mammary carcinoma was misinterpreted by FNAC as a ‘chondrosarcoma’. On review, this case showed scant cytologic material formed of pleomorphic malignant cells with enlarged, hyperchromatic nuclei in a myxoid background. Radiographic findings and a previous history of breast carcinoma were not available at the time of aspiration.
Primary malignant tumors
Follow-up of our cases confirmed that 34 cases (40%) were primary malignant neoplasms. The most frequent primary malignant neoplasm was osteosarcoma (Table 4). Smears were of low to moderate cellularity, featured pleomorphic malignant cells, and dispersed singly, forming small clusters and groups (Fig. 1a). These markedly anaplastic, rounded, and spindled-shaped cells had moderate to abundant cytoplasm and large pleomorphic hyperchromatic nuclei with occasionally prominent nucleoli. Atypical mitoses were abundant. Malignant giant cells (Fig. 1b), both mononuclear and multinuclear, were also frequent. Background showed faintly eosinophilic, amorphous aggregates of osteoid. In most cases of osteosarcoma (eight of 10), osteoid or osteoid-like material was appreciated along with pleomorphic cells, which rendered the diagnosis of osteosarcoma feasible. The smears of the chondroblastic variant of osteosarcoma (two of 10) showed an abundant chondromyxoid material in the background along with round to polygonal cells embedded in this material. This picture was similar to that encountered in high-grade chondrosarcoma.
Chondrosarcoma yielded gelatinous material on aspiration and was diagnosed cytologically in all six patients after correlation with the clinical and radiological information. Five cases were of low grade and one case was of high-grade malignancy. The low-grade chondrosarcomas showed mildly atypical chondroid cells situated in lacunae with an abundant chondromyxoid matrix. Dissociated single cells were relatively fewer in number. Mitotic figures were less frequently encountered. The high-grade tumors in contrast showed predominantly dissociated cells in a less abundant matrix. Cells were round to polygonal, with a variable amount of dense often vacuolated cytoplasm. Binucleate and multinucleate cells as well as mitotic figures were more frequent.
MFH smears (Fig. 1c and d) consisted mainly of a mixed population of spindle cells, histiocytoid, and pleomorphic cells. Varying amounts of multinucleated giant cells of the osteoclast type, as well as foamy cells and chronic inflammatory cells were observed. The nuclei of the tumor cells were quite atypical, especially these of the malignant giant cells. Typical and atypical mitoses were present.
The smears of Ewing sarcoma (Fig. 2) were cellular and showed malignant small round cells dispersed singly as well as arranged in loose clusters with frequent rosettes. The cells had scanty cytoplasm and round nuclei with fine granular chromatin and inconspicuous nucleoli. Five cases showed a positive reaction to CD99 and a negative reaction to LCA and desmin, confirming the diagnosis of Ewing sarcoma. A therapeutic decision was taken on the bases of FNAC only and neoadjuvant chemotherapy was started immediately without histopathological confirmation.
The smears from the three cases of plasma cell myeloma (Fig. 3) were composed of numerous plasma cells with varying degrees of maturation. Binucleate and multinucleate forms were frequently observed. All the cases showed an M band on serum electrophoresis. Chemotherapy started on the basis of FNAC diagnosis only. There was no need for biopsy for confirmation in these cases.
A case of chordoma (Fig. 4) was diagnosed in a 50-year-old man with a sacral tumor. Smears showed numerous large cells with pale-stained vacuolated cells and ovoid nuclei (physaliferous cells). Cells showed well-defined cytoplasmic borders, and the nuclei had evenly distributed chromatin with prominent nucleoli. Some smaller uniform tumor cells were also present.
Metastatic bone tumors
Fifteen cases (30.6%) proved to be metastatic neoplasms (Table 4 and Fig. 5). Nine cases (60%) had relevant radiological and clinical findings and the diagnosis was confirmed with IHC study. Surgical follow-up was available for the case mistyped as chondrosarcoma by FNAC. The most frequently aspirated sites showing metastatic bone tumor were the vertebral and pelvic bones. The most frequent type of metastatic carcinoma was adenocarcinoma (10 cases). In nine cases, the primary site was confirmed by an IHC panel. Three cases were positive for GCDFP-15, supporting a breast primary site of origin. Two cases showed a positive reaction for Cdx-2 and a negative reaction for GCDFP-15, supporting the colonic origin of tumor cells. In two cases, the tumor cells stained positively with TTF-1 and showed no IHC reaction for GCDFP-15 and Cdx-2, supporting pulmonary adenocarcinoma as primary. Two cases showed positivity to PSA. The remaining five cases that were not subjected to IHC because of deficient cytologic material, morphology together with the available history, confirmed the diagnosis of metastasis (two cases of squamous cell carcinoma of laryngeal and bladder origin, two cases of primary soft tissue sarcoma, and one case of malignant melanoma).
Suspicious and inadequate cases
We recommended histopathologic examination for all our suspicious and inadequate cases (13 cases, 15.3%). Subsequent histopathologic examination of the nine suspicious cases had confirmed their malignant nature (three metastatic carcinomas, four sarcomas, and two non-Hodgkin’s lymphoma). Core biopsies were performed for the four inadequate cases; their histopathologic examination confirmed two of them as malignant (osteosarcoma and Ewing sarcoma). The other two cases included one ‘giant cell tumor’ case and one ‘aneurysmal bone cyst’ case.
Benign, low-grade tumors, and inflammatory lesions
Twenty-three (27.1%) cases were benign or low-grade tumors (Table 1). A giant cell tumor was the most frequently identified lesion. The smears showed abundant round to oval polygonal or elongated mononuclear cells evenly mixed with numerous osteoclasts-like giant cells that may be very large and contain 50 to 100 nuclei. Radiographic findings (epiphyseal lytic lesion of long bones without perilesional sclerosis) and clinical characteristics (young adults) allowed for a definitive diagnosis in all five cases.
The smears from Langerhans cell histiocytosis cases were composed of Langerhans cells with its characteristic reniform nuclei with irregular notches and grooves. The Langerhans cells were frequently admixed with large number of eosinophils, as well as lymphocytes, neutrophils and plasma cells. Multinucleated osteoclast-like giant cells and occasionally lipid laden histiocytes were also seen. CD1a confirmed the diagnosis in three of the cases.
Three cases of aneurysmal bone cysts yielded rather low cellularity and considerable amount of blood. Spindle cell clusters and osteoclastic giant cells were observed along with hemosiderin-laden macrophages and osteoblasts. Radiologic features played a major role in arriving at the diagnosis.
The remaining benign cases were reported as negative for malignant cells with a comment statement describing the subcategory or subclassification of the lesion and the most likely differential diagnosis. The benign lipomatous lesions included classic lipoma, fibrolipoma, and angiolipoma. The inflammatory lesions included acute and chronic inflammation, abscess, and granulomatous inflammation.
The diagnosis of bone lesions can be made in a variety of ways, including FNAC, core biopsy, or open biopsy. Each of these diagnostic tools has advantages and disadvantages. When compared with open biopsy, FNAC is a simple, outpatient procedure that is well tolerated by patients and has a minimal risk of complications. When the material is sufficient, it is usually possible to separate benign from malignant lesions as well as to subtype malignancies. Differentiation between a primary and metastatic lesion can also be made using this method (Kabukçuoglu et al., 1998). In addition, the multiple direction of the FNAC needle enables sampling of different parts of large tumors, as opposed to a single small core biopsy or open biopsy. However, FNAC can have sampling errors attributable to low cellularity, inadequate sampling of the target, and copious cystic/bloody/necrotic material (Nagira et al., 2002). Despite these difficulties, FNAC is being used as a diagnostic modality for initial diagnoses, as well as for recurrences and metastases of bone lesions in numerous medical centers (Bommer et al., 1997; Söderlund et al., 2004; Handa et al., 2005; Mehrotra et al., 2007; Layfield, 2009). Treatment with radiation and/or chemotherapy can be initiated without any delay, as the aspiration wound is not endangered. In addition, using FNAC as the diagnostic method, the possibilities for salvage of a limb improve, because there is less disruption of soft tissue and less distortion of the affected bone (Jorda et al., 2000).
FNAC is considered an important procedure in the diagnosis of bone lesions because of its high accuracy. An accuracy rate of 91% was achieved in this study, but rates as high as 95% have been reported (Bommer et al., 1997; Jorda et al., 2000; Mehrotra et al., 2007). Adequacy of the aspirate plays an important role in achieving this rate, which in turn depends on the site, characteristics, histologic grade of the tumor, and adequacy of the clinical and radiologic data (Wahane et al., 2007).
As many malignant primary bone tumors have palpable soft tissue extensions, which can be easily aspirated, we used size 23 G needles for most of the aspirates. Needles of shorter lengths only hit the reactive zone and may lead to false-negative results. In the deeply seated lesions, the use of image guidance can help to localize the lesion appropriately. The material obtained was fixed immediately in 95% ethyl alcohol and stained using the modified Papanicolaou method. This has the advantage of better nuclear detail and ease of comparison with histological sections. Whenever sufficient material is available, cell block preparations can be processed and immunocytochemistry can be applied; this helps to resolve morphologic deadlocks.
As reported elsewhere (Nnodu et al., 2006; Eyre et al., 2009; Layfield, 2009), osteosarcoma was the most frequent primary malignant tumor. The clinical features of age at presentation, rapid onset, radiographof an irregularly outlined osseous defect with cortical destruction, sun-burst appearance, and Codman’s triangle with the characteristic cytological features mentioned previously (Fig. 1a and b) were mandatory and enough for establishing the diagnosis. All cases of osteosarcoma (n=10) were diagnosed by these collaborative studies and were confirmed by histopathology after excision of the tumors. In one case, the morphologic features were diagnostic of malignancy; however, typing as osteosarcoma was not conclusive because of the absence of osteoid.
It was difficult to distinguish our two cases of the chondroblastic variant of osteosarcoma from high-grade chondrosarcoma. This distinction was possible only as patients were young (12 and 16 years old) together with metaphyseal involvement of the affected bones and relatively more nuclear hyperchromasia and pleomorphism.
The second most frequent primary malignant bone tumor in our study was Ewing sarcoma (eight of 34 cases), constituting 23.5% of the primary malignant cases. The reported incidence in the literature varies widely from 6% (Chow et al., 2011) to 39% (Eyre et al., 2009). This may depend on differences in the age groups involved in each study. The predilection for the teenage group, together with the characteristic radiographic findings of a permeative diaphyseal process associated with an aggressive ‘onion-skin’ type of periostitis, suggests the diagnosis of Ewing sarcoma. Unfortunately, cytomorphologic features cannot differentiate Ewing sarcoma from other blue round cell tumors of childhood (lymphoma mainly). Accordingly, more studies were needed for confirmation (immunocytochemistry on cell blocks, or tissue biopsy and IHC). The cytologic diagnosis of Ewing sarcoma was confirmed by an immunocytochemical panel in 62.5% of cases (positive reaction to CD99 and negative reaction to LCA). In the remaining 37.5% of cases, tissue biopsy and IHC confirmed the diagnosis.
In the current study, cases of chondrosarcoma constituted 17.6% of all primary tumors. This is comparable to 20% reported by Flemming and Murphey (2000). All the six cytologically diagnosed cases of chondrosarcoma were confirmed histopathologically. One case was diagnosed cytologically as chondrosarcoma and was proved to be metastatic lobular carcinoma by histopathology. This case lacked adequate clinical data including a history of primary breast cancer and also the radiological findings of the bony lesion were insufficient. This deficient clinicoradiologic correlation together with the subtle morphologic features of tumor cells that present as separate round and oval cells with mild atypical nuclear features and focal cytoplasmic vacuolations resulted in false cytologic typing of this case as chondrosarcoma.
Plasma cell myeloma as an entity was found in 8.8% of our studied cases of primary bone tumors. This result is in agreement with 8.3% observed by Shah et al (1999). Cytological smears of all three cases showed typical plasma cells so that a correct diagnosis could be made in all of the cases. Serum electrophoresis was useful in confirming the diagnosis.
Excellent cytologic details obtained by FNAC in MFH and the accuracy of the results make FNAC a highly reliable procedure in the diagnosis of this tumor (Tarkkanen et al., 2006). Chordomas (Walaas and Kindblom, 1991; Bommer et al., 1997) and NHL (Chao et al., 2001) can also be diagnosed easily using this diagnostic technique.
Metastatic tumors accounted for 17.6% (15 of 85 cases) of bone lesions in this study, which is less than the figures reported in the literature: 44 and 28.1% (Jorda et al., 2000; Nnodu et al., 2006). This could be explained by the fact that in our institute, radiology alone is considered sufficient for the diagnosis of metastatic tumors in bone in patients with a history of primary tumor elsewhere. Previous studies have adequately reported the high diagnostic accuracy of FNAC of bone in the diagnosis of metastatic lesions (James and Frable, 1983; Jorda et al., 2000). In our series, 93.3% (14 of 15 cases) of metastatic bone tumor were diagnosed correctly by FNAC. The presence of a clinical history of malignancy and the familiarity with the previous morphology of the known malignancy enhance the ability of the FNA to make a more definitive diagnosis, which may explain why 14 of the 49 malignancies were successfully subclassified as a metastatic carcinoma. An appropriate diagnosis of a metastatic lesion by FNAC facilitates either nonoperative management or contemporary surgical reconstructive techniques.
Benign and borderline bony lesions constituted 23 of 85 cases of our study, with a frequency of 27%. This result is somewhat lower than the 38% reported by Moatasim and Haque (2005). Giant cell tumor of bone and inflammatory bony lesions constituted the majority of these cases, with a frequency of 21.7% each. The frequency of cases of giant cell tumor in this study was slightly lower than the 28% reported in the literature (Eyesan et al., 2011). This may be attributed to the relatively larger number of cases in our study.
The characteristic radiological picture of giant cell tumor plays a crucial role in the diagnosis of this borderline tumor. This tumor presents as a lytic lesion centered in the epiphysis but involving the metaphysis and extending at least in part to the adjacent articular cortex. Most are eccentric, but become symmetric and centrally located with growth. Most cases show circumscribed borders or so-called geographical destruction with no periosteal reaction unless a pathological fracture is present. There is no mineralized tumor matrix (Purohit and Pardiwala, 2007). This characteristic radiological presentation together with the cytomorphologic features previously mentioned led to the diagnosis of giant cell tumor in our five cases studied, with histopathologic confirmation in four of them. All the five cases studied showed no atypia or suspicion of malignancy.
The differential diagnosis between osteomyelitis and neoplasm may be difficult clinically and radiologically; therefore, the results of bacterial culture and the cytologic presence of mostly inflammatory cells help to establish the diagnosis of osteomyelitis (Nnodu et al., 2006). Inflammatory bony lesions in the current study included two cases of acute and three cases of chronic osteomyelitis.
The rest of our cytologically diagnosed ‘benign lesions’ included four cases of Langerhans cell histiocytosis, three of lipomatous lesion, three of hemangioma, and three of aneurysmal bone cyst, with frequencies of 17.4 and 13% (for the latter three tumors), respectively.
The more problematic group is the group of inadequate FNAs. There are no established adequacy criteria for bone cytology. Thus, the number of inadequate cases varies between cytopathologist, by institution, and by study. One study defined adequacy in bone FNA biopsies as the presence of at least five clusters of 10 unobscured cells on the majority of slides (Kreicbergs et al., 1996). Acceptance of FNAC of bone as a diagnostic technique has been impeded by the inability to obtain adequate smears. However, the rate of inadequate aspirates, ranging from 0% (Layfield et al., 1987) to 33% (Dollahite et al., 1989), compares favorably with the rate published for open or cutting core needle biopsies (Springfield and Rosenberg, 1996; Gogna et al., 2008). The presence of a cytopathologist during the FNAC sampling procedure should reduce the percentage of inadequate aspirates. Thus, immediately after an unsuccessful FNAC, the radiologist could proceed to obtain a core biopsy (Ayala et al., 1995). In this study, an experienced cytopathologist usually attended the procedure of FNAC, which can partially explain our low rate (4.7%) of initial insufficient aspirates. The variability in reported inadequacy rates also depends on the number of cases reviewed in the studies. In fact, the highest reported inadequacy rate was reported in a study with no onsite evaluation, where 97 of 314 cases (31%) were deemed inadequate (Jorda et al., 2000). Thus, our study and other studies (Bommer et al., 1997; Jorda et al., 2000; Nnodu et al., 2006; Layfield, 2009) suggest that onsite evaluation not only reduces the inadequacy rate but also significantly enhances the diagnostic value of FNA in bone cases.
We conclude that when sampling is adequate and the clinicoradiologic findings are available, FNAC of bone is a highly accurate diagnostic technique. Inflammatory conditions, benign, nonfibrotic bone lesions as well as primary and metastatic malignant tumors can be diagnosed correctly. Considering the overall advantages and cost analysis, FNAC may be suggested as the initial method of choice for the evaluation of bony lesions in most clinical settings, especially in resource-challenged countries.
Conflicts of interest
There are no conflicts of interest.
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