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The Detection of Isolated Tumor Cells in Bone Marrow Comparing Bright-Field Immunocytochemistry and Multicolor Immunofluorescence

¹ Department of Surgery, University of Vermont College of Medicine, Given Building, 89 Beaumont Avenue, Burlington, Vermont 05405
² Charleston Area Medical Center Institute, 3110 MacCorkle Avenue SE, Charleston, West Virginia 25304
³ Department of Surgical Pathology, Fletcher Allen Health Care, 111 Colchester Avenue, Burlington, Vermont 05405
⁴ Department of Orthopaedics and Rehabilitation, University of Vermont College of Medicine, 430A Stafford Hall, 95 Carrigan Drive, Burlington, Vermont 05405

Correspondence: Address correspondence and reprint requests to: David N. Krag, MD, FACS; E-mail: david.krag{at}uvm.edu.

Background: The detection of isolated tumor cells in bone marrow by immunocytochemistry (ICC) has been reported to predict progression of early-stage breast cancer. The most common staining procedure uses bright-field ICC with cytokeratin (CK) antibodies to label isolated tumor cells. However, this method can result in false-positive staining events. We used multicolor immunofluorescence (IF) to develop a more specific assay for detecting isolated tumor cells in marrow samples from breast cancer patients.

Methods: We compared ICC and IF side by side for detection of cancer cells and false-positive staining events on bone marrow aspirates from breast cancer patients, bone marrow from healthy donors, and healthy donor blood spiked with cancer cells. The primary target for isolated tumor cell detection was CK for both methods. IF used an additional set of antibodies to label hematopoietic cells (HCs).

Results: The detection rate of CK⁺ events in breast cancer patient bone marrow aspirates was 18 (58%) of 31 for ICC and 21 (68%) of 31 for IF. However, with IF, 17 of 21 CK⁺ cases were stained with HC markers and thus were identified as false-positive events. A surprisingly high CK⁺ event rate was observed in healthy donor blood and marrow. In all healthy donor samples, CK⁺ events were readily identified as HCs by IF. Detection sensitivity of spiked cancer cells in donor blood was similar for both methods.

Conclusions: There is a high frequency of CK⁺ events in blood and marrow, and it is important to note that this is observed both in patients with and those without cancer. IF with multiple HC markers allows straightforward discrimination between CK⁺ cells of hematopoietic and nonhematopoietic origin.

Key Words: Immunocytochemistry • Bone marrow • Cytokeratin • Immunofluorescence

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