57�C62 More recently, high throughput technologies have Tofacitinib baldness made major contributions to the study of self-antigen�Cantibody systems as serologic biomarkers in osteosarcoma.10,11 In this study, we have used a novel ELISA to evaluate the presence of circulating ANG�CIgM immune complexes in the peripheral blood sera of patients with osteosarcoma and healthy individuals, and also to evaluate the usefulness of ANG�CIgM detection for an early diagnosis of osteosarcoma. Materials and Methods Patients The study included 117 patients with newly diagnosed osteosarcoma in the Department of Bone Tumors of the National Institute of Rehabilitation, Mexico City, Mexico between January 2007 and September 2009; their mean age was 23.8 years (range: 4�C74 years), and 76 men and 41 women took part.
Tumor staging was based on radiography, computed tomography, operative findings and pathology reports in accordance with the Enneking Surgical Staging System. All patients had histopathologic confirmation of osteosarcoma according the WHO classification. The median follow-up time was 43 months (range: 24�C72 months). Individual patient records were traced where possible. The comparative group consisted of 117 patients with other tumors (osteocondroma, fibrous displasia, encondroma, condroblastoma, Ewing��s tumor, giant cell bone tumor, desmoplastic fibroma, chondromyxoid fibroma, simple bone cyst, aneurysmal bone cyst, Langerhans�� cell histiocytosis) matched for age (��5 years), gender and ethnicity in the Department of Bone Tumors of National Institute of Rehabilitation, Mexico City, Mexico.
Control subjects The general reference (normal) control samples consisted of 117 healthy individuals (75 men and 42 women with a mean age of 25.2 years; range: 5�C73 years) in the Blood Transfusion Service of the National Institute of Rehabilitation. The absence of disease was confirmed by physical examination, clinical history and routine laboratory tests. Blood sampling Seven milliliters of the peripheral venous blood was drawn into a serum separator tube (Vacutainer Systems, code 607213, Becton-Dickinson, USA). Blood was allowed to clot for 1 h at room temperature (RT). Sera was obtained after centrifugation at 3000 r.p.m. for 10 min at 4 ��C. All serum samples were stored in 300 ��L aliquots at ?80 ��C until analysis.
Peripheral venous blood samples were collected from cancer patients before or after surgery, and during antiangiogenic therapy. Reagents All reagents were of analytical grade and were obtained from Sigma�CAldrich Ltd, Poole, UK, unless otherwise indicated. Gel-filtration Serum samples from 117 patients with biopsy-proven osteosarcoma were grouped and subjected to gelfiltration AV-951 analysis. One hundred microliters of pooled sera were analyzed as previously described. The presence of ANG�CIgM and ANG in the fractions collected from the gel-filtration column every 30s were tested by ELISA.